JP2001526025A - Method for preparing vaccine against cancer - Google Patents

Abstract

  (57) [Summary] The present invention relates to methods of preparing immunogenic, prophylactically and therapeutically effective conjugates of heat shock proteins non-covalently bound to antigenic peptides of cancer cells. The method of the present invention comprises constructing a cDNA library from cancer cell or pre-neoplastic cell RNA, expressing the cDNA library in a suitable host cell, and recovering the immunogenic complex from the cell. Including. A large amount of the immunogenic complex can be obtained by performing a large-scale culture of a host cell containing the cDNA library. The conjugate can be used as a vaccine to elicit a specific immune response against cancer or pre-neoplastic cells, or to treat or prevent cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This invention was made with government support under Grant No. CA44786 approved by the National Institutes of Health and Grant No. N652369715812 approved by the United States Navy. The government has certain rights in the invention.

1. Introduction The present invention relates to a method for preparing a large amount of an immunogenic substance useful as a vaccine for preventing and / or treating cancer. The vaccine consists of a non-covalent complex of heat shock proteins (hsp), including but not limited to hsp70, hsp90, gp96, and protein disulfide isomerase, and an antigenic peptide. A vaccine can elicit or enhance a subject's immune response to certain cancers.

2. BACKGROUND OF THE INVENTION 2.1. Pathology and Biology of Cancer Cancer is characterized primarily by an increase in the number of abnormal cells from a given normal tissue. The process of disease also involves the invasion of adjacent tissues by these abnormal cells and the spread of these abnormal cells via the circulatory system to local lymph nodes and distant sites (metastasis). Clinical data and molecular biology studies indicate that cancer is a multi-step process beginning with small pre-tumor changes that can progress to neoplasia under certain conditions.

[0004] Precancerous abnormal cell growth is exemplified by hyperplasia, dysplasia, or most strictly dysplasia (for a review of such abnormal growth states, see Robbins and Angell, 197
6, Basic Pathology, 2nd edition, WB Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled cell growth that involves an increase in the number of cells in a tissue or organ without significant changes in structure or function. By way of example only, endometrial hyperplasia often precedes endometrial cancer. There is a metamorphosis 1
A form of controlled cell growth in which one type of adult cell or a fully differentiated cell is replaced by another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Exceptional metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is often a precursor to cancer, found primarily in the epithelium, and is the most disorderly form of non-neoplastic cell proliferation, with loss of individual cell homogeneity and loss of structural orientation of cells . Dysplastic cells often have abnormally large, deeply stained nuclei and exhibit polymorphism. Dysplasia characteristically occurs at sites where chronic irritation or inflammation is present, and is often found in the neck, respiratory tract, oral cavity and gall bladder.

[0005] Neoplastic lesions clonally form and may increasingly enhance the ability to invade, proliferate, metastasize and heterogeneize, especially under conditions where tumor cells escape the host's immune surveillance mechanism (Roitt). , I., Brostoff, J and Male, D., 1993, Immunology, Third Edition.
, Mosby, St. Louis, pps. 17.1-17.12).

2.2. Vaccination Vaccination has eradicated certain diseases such as polio, tetanus, varicella and measles in many countries of the world. This method has exploited the ability of the immune system to prevent infectious diseases. Vaccination with non-live substances, such as proteins, generally causes an antibody response or a CD4 + helper T cell response (Raychaudhuri and Morrow, 1993, Immunology Today, 14: 344-348). On the other hand, vaccination or infection with live substances, such as live cells or infectious viruses, generally causes a CD8 + cytotoxic T-lymphocyte (CTL) response. The CTL response is critical for protection against cancer, infectious viruses and bacteria. This causes implementation problems.
Because the only way to achieve a CTL response is to use a live substance that is itself pathogenic. This problem is usually circumvented by using attenuated virus strains and strains or by killing all cells that can be used for vaccination. While these strategies have worked, the use of attenuated strains always carries the danger that the attenuated material may genetically recombined with host DNA and become a toxic strain. I have. Therefore, there is a need for a method that can induce a CD8 + CTL response by vaccinating with a non-live substance such as a protein in a specific manner.

[0007] In the age of tumor immunity, the antigens on methylcholanthrene (MCA) -induced sarcomas were tumor-specific, incapable of detecting these antigens in normal mouse tissues by transplantation assays An experiment by Prehn and Main (Prehn et al., 1957, J.
Natl. Cancer Inst. 18: 769-778). This view was confirmed by further experiments demonstrating that tumor-specific resistance to MCA-induced tumors could be induced in the mice that originated the tumors (Klein et al., 1960, Cancer Res. 20: 1561).
-1572).

In subsequent experiments, tumor-specific antigens were also found in tumors induced by other chemical or physical carcinogens, or in spontaneous tumors (Kripke, 1974, J.
Natl. Cancer Inst. 53: 1333-1336; Vaage, 1968, Cancer Res. 28: 2477-2483;
Carswell et al., 1970, J. Natl. Cacer Inst. 44: 1281-1288). In these studies,
These antigens are also commonly referred to as "tumor-specific transplantation antigens" or "tumor-specific rejection antigens" because protective immunity against the growth of transplanted tumors was used as a basis for tumor-specific antigens. Several factors can significantly affect the immunogenicity of tumors, including, for example, the specificity of the carcinogen involved, the immune competence of the host, and the latency period (Old et al., 1962, Ann. NY Acad. Sci. 101: 80-106; Bartlett, 1972, J. Nat.
l, Cancer Inst. 49: 493-504).

[0009] Most, if not all, carcinogens are mutagens capable of causing mutations, which lead to the expression of tumor-specific antigens (Ames, 1979, Science 204: 587-59).
3; Weisburger et al., 1981, Science 214: 401-407). Some immunosuppressive carcinogens
(Malmgren et al., 1952, Proc. Soc. Exp. Biol. Med. 79: 484-488). Experimental evidence suggests that there is a certain opposite correlation between tumor immunogenicity and latency (time between tumor exposure and carcinogen exposure) (Old et al., 1962, Ann. . NY
Acad. Sci. 101: 80-106; and Bartlett, 1972, J. Natl. Cancer Inst. 49: 493-5.
04). Other studies have revealed the presence of tumor-specific antigens that do not lead to rejection, but nonetheless can potentially stimulate specific immune responses (Roit
t, I., Brostoff, J. and Male, D., 1993, Immunology, 3rd ed., Mosby, St.
Louis, pp. 17.1-17.12).

2.3. Heat Shock Protein Heat shock protein (hsp) is synonymous with stress protein. The first stress proteins identified were proteins synthesized by cells in response to heat shock. To date, three large families of hs based on molecular weight
p has been identified. This family includes hsp60, hsp70, hsp90
The numbers reflect the approximate molecular weight (in kilodaltons) of the stress protein. Many members of these families have been found to be induced in response to other stressful stimuli, such as undernutrition, metabolic destruction, oxygen radicals, and infection by intracellular pathogens. (Welch, May 1993, Scientific American
56-64; Young, 1990, Annu. Rev. Immunol. 8: 401-420; Craig, 1993, Scienc.
e 260: 1902-1903; Gething et al., 1992, Nature 355: 33-45; and Lindquist et al.
1988, Annu. Rev. Genetics 22: 631-677).

[0011] The major hsp is accumulated at high levels in stressed cells, but is present at low to moderate levels in unstressed cells. For example, the highly inducible mammalian hsp70 is undetectable at normal temperatures but becomes one of the most actively synthesized proteins in heat shocked cells (Welch et al., 1985,
J. Cell. Biol. 101: 1198-1211). In contrast, hsp90 and hsp6
The 0 protein is abundant at normal temperatures in most, if not all, mammalian cells and is further induced by heat (Lai et al., 1984, Mol. Cell. Biol. 4:
2802-2810; van Bergen en Henegouwen et al., 1987, Genes Dev. 1: 525-531).

[0012] Studies of cellular responses to heat shock and other physiological stresses have shown that not only cellular defenses against their adverse conditions, but also essential biochemical and immunological processes in unstressed cells. It became clear that hsp was involved. This hsp performs different kinds of chaperoning functions. For example, hsp70, located in the cytoplasm, nucleus, mitochondria, or endoplasmic reticulum, (Lindquist, S. et al., 1988, Ann. Rev. Genetics 22: 631-677) is involved in the presentation of antigen to cells of the immune system. And is also involved in the movement, folding and assembly of proteins in normal cells. Hsps can bind proteins or peptides and can release bound proteins or peptides in the presence of adenosine triphosphate (ATP) or at low pH.

[0013] Other stress proteins involved in protein folding and assembly include, for example, protein disulfide isomerase (PDI), which catalyzes disulfide bond formation, isomerization, or ER reduction (Gething et al.). , 1992, Na
ture 355: 33-45).

[0014] Heat shock proteins are among the most highly conserved proteins in existence. For example, DnaK, hsp70 from E. coli has about 50% amino acid sequence identity with the hsp70 protein from excoriate (Bardwell et al., 1984, Proc.
tl. Acad. Sci. 81: 848-852). Also, the hsp60 and hsp90 families similarly show high levels of intra-family conservation (Hickey et al., 1989, Mol. Cell. B.
iol. 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol. 9: 2279-2283). in addition,
The hsp60, hsp70 and hsp90 families have, for example, 35%
It has been discovered that proteins that are related to stress proteins in sequences with greater amino acid identity, but whose expression levels are not altered by stress.

2.4. Immunogenicity of the heat shock / stress proteins hsp70, hsp90 and gp96 Srivastava et al. Have shown an immune response to methylcholanthrene-induced sarcomas in inbred mice (1988, Immunol. Today 9:78). -83). In these studies, it was found that the molecules responsible for the different immunogenicity of these tumors were identified as a 96 kDa cell surface glycoprotein (gp96) and an 84-86 kDa intracellular protein (Srivastava, PK et al., 1986, Proc. Natl. Acad. Sci. USA 83: 3407
-3411; Ullrich, SJ et al., 1986, Proc. Natl. Acad. Sci. USA 83: 3121-3125). Immunization of mice with gp96 or p84 / 86 isolated from a particular tumor, the mice were immunized against that particular tumor but not against antigenically different tumors . Isolation and characterization of the genes encoding gp96 and p84 / 86 show significant homology between them, with gp96 and p84 / 86 being equivalent to the endoplasmic reticulum and cytosol of the same heat shock protein, respectively. (Srivastava, PK et al., 1988, Immunogenetics 28: 205-207; Srivastava,
PK et al., 1991, Curr. Top. Microbiol. Immunol. 167: 109-123). In addition, hsp7
A value of 0 indicates that it elicits immunity against the tumor from which it was isolated, but not against an antigenically distinct tumor. However, peptide-depleted hsp70 was found to lose its immunogenic activity (Udono, M. and Srivasta
va, PK, 1993, J. Exp. Med. 178: 1391-1396). These observations indicate that these heat shock proteins, not themselves immunogenic, form a non-covalent complex with the antigenic peptide, and the complex is specific for the antigenic peptide. (Srivastava, PK, 1993, Adv.
Cancer Res. 62: 153-177; Udono, H. et al., 1994, J. Immunol., 152: 5398-5403;
Suto, R. et al., 1995, Science, 269: 1585-1588).

The use of non-covalent complexes of stress proteins and peptides purified from cancer cells to treat and prevent cancer is described in PCT Publication WO 96/1041 dated April 11, 1996.
1, and WO 97/10001 dated March 20, 1997. (Srivast respectively
Filed February 7, 1997 by Ava and Chandawarkar, February 1997 by Srivastava
Co-pending U.S. Patent Applications 08 / 796,319 and 08 / 796,31 filed on the 7th.
See also Issue 6. Each of which is incorporated herein by reference in its entirety). Stress protein-peptide complexes can be isolated from cells infected with the pathogen and treat or prevent infections caused by pathogens such as viruses and other intracellular pathogens including bacteria, protozoa, fungi, and parasites. Can also be used. See PCT Publication WO 95/24923 dated September 21, 1995. The immunogenic stress protein-peptide complex binds stress proteins and antigenic peptides in vit
It can also be prepared by conjugation with ro. The treatment and prevention of cancer and infectious diseases using such a conjugate is described in PCT Publication WO 97/9897, Mar. 20, 1997.
/ 10000. Using stress protein-peptide complex, in v
The sensitization of antigen-presenting cells with itro for use in adoptive immunotherapy is described in PCT Publication WO 97/10002, dated March 20, 1997.

[0017] Purification of the stress protein-peptide complex was described previously;
See PCT Publication WO 95/24923 dated September 21. The amount of immunogenic material obtained for use in preparing a vaccine against cancer is directly related to the amount of starting cancer cells. The supply of cancer cells to produce hsp-peptide conjugates is often very limited, as the number of cancer cells obtained from subjects is small, especially when the cancer is in its early stages. Although some types of cancer cells can be cultured in vitro, they are less preferred than using complexes known as representatives of cancer cells in vivo. For commercial production of vaccines or therapeutics, it is advantageous to constantly supply large amounts of the hsp-peptide conjugate. Thus, there is a need for a reliable long-term source of hsp-peptide conjugates that does not depend on the availability of fresh cell samples from cancer patients. The method of the present invention does not rely on the large or continuous procurement of such cancer cells from a subject, even when only a small amount of tumor tissue is available from the patient for use. -Can be used to provide a peptide conjugate.

3. SUMMARY OF THE INVENTION The present invention relates to a method for increasing the production of an immunogenic substance that can be used for the prevention and treatment of cancer. The immunogenic composition prepared by the method of the present invention comprises a non-covalently bound molecular complex of a heat shock protein (hsp) and an antigenic (or immunogenic) peptide. The complex prepared by the method of the present invention was produced intracellularly in a complex comprising hsp from a selected recombinant host cell and an antigenic peptide expressed from cDNA of a cancer cell; The antigenic peptides of the complex are representative of the antigenic peptides found in such cancer cells. The present invention creates a cDNA library from cancer cells and uses the cDNA library to construct a recombinant DN.
A method for producing an immunogenic hsp-peptide complex in a host cell by Method A, which immunizes an individual who receives the complex with the cancer cell.

Generally, the methods of the present invention involve obtaining (eg, isolating) cancer cells from one or more individuals, preparing RNA from the cancer cells, producing cDNA from the RNA, and hosting the cDNA in a host. Transfecting the cells into cells, culturing the host cells so that the cancer-derived cDNA is expressed, and purifying the heat shock protein-peptide complex from the host cells.

[0020] cDNA prepared from cancer cell RNA is referred to herein as "cancer cDNA", which cDNA is optionally amplified prior to introduction into a host cell for expression. The cDNA is optionally inserted into a cloning vector prior to expression for replication purposes. The cDNA is inserted into an expression vector or integrated into a chromosome and operably linked to control elements, such as a promoter, to express the encoded protein in vitro in a suitable host cell. . The cDNA is introduced into the host cell where it is expressed, thereby producing a non-covalent complex of hsp and the peptide (including the peptide encoded by the cancer cDNA) in the cell. Recombinant host cells can be cultured on a large scale for large-scale production of immunogenic complexes. Cancer cDNA libraries can be stored for future use (e.g., by lyophilization or freezing) or in a cloning vector in a suitable host cell to address the increasing demand for immunogenic complexes. Volume can be increased by replication.

An immunogenic composition prepared from a host cell expressing a cancer cDNA consists of a host cell hsp non-covalently linked to a peptide, which peptide specifically obtained the RNA. It is a peptide derived from cancer cells. Such a conjugate can elicit an immune response in a patient against cancer cells, the response being therapeutically or prophylactically effective. Preferably, the patient is a subject who has obtained the cancer cells used to produce the cDNA. Alternatively, cancer cells can be obtained from one or more subjects different from the patient but having a cancer of the same tissue type (eg, gastric cancer, breast cancer, colon cancer, lung cancer, etc.).

Optionally, the host cell for expression of the cancer cDNA is genetically engineered to co-express one or more hsp genes by genetic recombination, such that the immunogenic peptide binds non-covalently to the hsp. It is possible to increase the production of a complex including the above. Certain compositions of the present invention and methods for their preparation are described in the sections and subsections below.

4. BRIEF DESCRIPTION OF THE FIGURES (See below for description of figures ) 5. Detailed Description of the Invention The present invention provides for the preparation of immunogenic compositions that can be used for the prevention and treatment of cancer. It is intended to apply recombinant DNA technology to The immunogenic composition prepared by the method of the present invention comprises a heat shock protein (hsp) and an antigenic peptide that is present as a peptide in cancer cells or is part of a protein that is present in cancer cells. A non-covalently bound molecular complex. Such hs
The p-peptide complex can induce a specific immune response against cancer cells in mammals.

[0024] The immunogenic hsp-peptide conjugate is produced naturally in cancer cells. Because it is not always possible to obtain large numbers of cancer cells, the amount of hsp-peptide conjugates available from cancer cells is sometimes very limited. Therefore, it is an object of the present invention to address the potential problem of limited supply of starting cellular material to produce large amounts of the hsp-peptide complex in recombinant host cells. Another object of the present invention is to provide a method for preparing a cDNA library from cancer cells.

In one embodiment, the present invention provides a method of making a cDNA library (hereinafter referred to as a “cancer cDNA library”) from RNA of a cancer or pre-neoplastic cell, and an antigenic peptide of the cancer or pre-neoplastic cell For producing an immunogenic composition comprising a non-covalent complex of a protein and a heat shock protein of a host cell using the cancer cDNA library. It will be clear that the method of the present invention is also applicable using pre-neoplastic cells, but the present invention refers to "cancer cells" when describing use for the prevention or prevention of cancer. Described in terms. The hsp-peptide complex is recovered from a culture of the recombinant host cell and preferably purified. An example of the hsp-peptide complex production method of the present invention is shown in FIG.

In certain embodiments, the present invention relates to cDNs made from RNA molecules of cancer cells.
A molecule is introduced into one or more host cells, where each cDNA molecule has at least one cDNA
Operably linked to or associated with a control region that regulates the expression of a DNA molecule; a host cell that contains the cDNA is treated with a protein (including a peptide) encoded by the cDNA molecule. Culturing under conditions such that Hsp is expressed in the host cell; and recovering a complex in which the heat shock protein non-covalently binds to one or more peptides from the host cell. Methods of production are provided.

[0027] In yet another specific embodiment, the invention relates to a method of producing a host cell comprising an expression construct comprising a cDNA encoding a cancer protein (eg, a peptide), wherein the cancer protein is expressed in the host cell. And a method for producing an hsp-peptide complex, which comprises culturing to bind non-covalently to hsp of a host cell and recovering the hsp-peptide complex.

The hsp-peptide complex recovered from the recombinant host cells can be purified to obtain a high purity of the complex. In certain embodiments, the conjugate can be purified to apparent homogeneity using the methods described in Section 5.2.

[0029] As used herein, the preneoplastic cells include antigenic cells which are infected with a carcinogenic infectious agent such as a virus, but have not yet become tumor cells.
Or antigenic cells exposed to mutagens or carcinogenic agents such as DNA damaging agents, radiation and the like. Other cells used to create cDNA libraries include preneoplastic cells that are in a transition from normal to tumor morphology, characterized by morphological, physiological, or biochemical functions. Can be Preferably, the cancer cells and pre-neoplastic cells used in the method of the invention are of mammalian origin. Mammals encompassed by this aspect of the invention include humans, companion animals (e.g., dogs and cats), livestock (e.g., sheep, cows, goats, pigs, and horses), and experimental animals (e.g., mice, rats, and rabbits) ), And wildlife captured or in nature.

[0030] It is estimated that about 30,000 to 120,000 different messenger RNA (mRNA) species are present in normal mammalian cells in their cytoplasm (Bishop et al., 1974, Nature
250: 199-204; Ryffel et al., 1975, Biochem. 14: 1379-1385). In various embodiments, a cancer cDNA library of the invention comprises at least 30,000, at least 60,000, or at least 90,000 independent cDNAs. 1 for all different RNA species
To properly represent one cDNA library, a cancer cDNA library (for example, when made from total mRNA) has at least one more cDNA than the estimated number of mRNA species, for example, 300,000 cancer cDNA libraries. From 1,200,000 independent different cds
Those containing the NA clone are preferred.

The cancer cDNA library of the present invention comprises a pool of cDNAs made from cancer cell RNA, preferably total poly A + RNA (mRNA). Preferably, each of the isolated cancer cDNAs is operably linked to at least one control region (eg, a promoter) that regulates expression of the cDNA in a suitable host cell or organism. Alternatively, the cDNA is inserted at a position in the chromosome so that the cDNA is operably linked to at least one control region that regulates expression of the cDNA in the host cell or organism. In order to facilitate homologous recombination inside the host cell,
Can be adjacent.

A library of expression or replicable constructs containing cancer cDNAs may be used in vitro.
(If desired), aliquoted, and lyophilized or frozen as nucleic acid molecules for future use. To meet the increasing demand for hsp-peptide complexes, the library can be thawed and introduced directly into host cells for hsp-peptide complex production. Alternatively, the library is hsp-
Prior to introduction into a suitable host cell for production of the peptide complex, it can be cloned in intermediate cells and / or expanded by replication in a cloning vector. In fact, cancer cDNA libraries capture and preserve antigenic genetic material that is actively expressed in cancer cells.

An expression construct or expression vector containing a cancer cDAN can be introduced and maintained in a host cell by any method known in the art. The cancer cDNA of a cancer cell, if properly transcribed, translated, and processed to produce the cancer cell protein or peptide in the host cell or host organism, some of which are antigenic and stressful When complexed with a protein, an immune response can be induced. The term "cancer cDNA host cell" as used herein means a host cell that contains a cancer cDNA.

Expression of a cancer cDNA in a recombinant host cell produces one or more proteins (eg, peptides) of the cancer cell and fragments thereof, which bind to non-covalently bound host cell stress proteins. Form a complex. Because some of the cancer cell's proteins are antigenic / immunogenic, the peptide / protein that forms a complex with the hsp is specifically immunized against the cancer cells in which the peptide / protein is present in the host. (See PCT Publication WO 96/10411, dated April 11, 1996). The immunogenicity of such compositions can be tested by methods known in the art and as described in Section 5.3. Such a non-covalent complex of a cancer peptide and hsp can be used as a vaccine to treat or prevent the type of target cancer from which the antigenic cancer peptide originated. Thus, recovering or purifying an immunogenic composition useful as a vaccine from a culture of recombinant host cells expressing a cancer cDNA and producing a non-covalent complex of a cancer peptide and hsp. Can be.

Depending on demand, recombinant host cells containing the cancer cDNA library can be pooled and / or taken up; or expanded to increase the number of clones containing the library Or can be frozen and stored in liquid nitrogen, so that the batch of recombinant host cells can be revived in the future and used multiple times. By culturing cancer cDNA library cells continuously or in batches on a suitable large scale, antigenic peptides expressed in cancer cells can be produced in large amounts in recombinant host cells. Desirable immunogenic compositions useful for the treatment and prevention of cancer, including non-covalent complexes of host cell hsps and cancerous cell antigenic proteins / peptides, can be used for large-scale serialization of cancer cDNA host cells. It can be prepared or purified from a culture or batch culture. Thus cancer
cDNA libraries provide an unaltered, reproducible, and rich source of useful immunogenic compositions.

In various embodiments, any cancer cell, preferably a human cancer cell, can be used in the methods of the invention to generate a cancer cDNA library. The cancer cells provide RNA encoding a protein expressed in the cancer cells. Cancers that can be treated or prevented with the immunogenic compositions prepared by the methods of the present invention include, but are not limited to, tumors such as sarcomas and carcinomas.
Examples of cancers susceptible to the methods of the present invention are listed in Section 5.4.

In one embodiment of the invention, tissues or cells isolated from cancer, including pre-neoplastic lesions, cancer that has spread to multiple distant sites, can be used in the methods of the invention. For example, cells found in abnormally proliferating tissues, leukemia cells in the bloodstream, metastatic foci, and solid cancer tissues can be used.

In another embodiment, a cell line derived from a pre-neoplastic lesion, a cancer tissue, or a cancer cell, wherein the cells of the cell line also have at least one antigenic determinant common to an antigen on the target cancer cell If it has the above, it can be used. Preferred are cancerous tissues, cancerous cells, cells infected with a carcinogenic agent, other pre-neoplastic cells, and cell lines of human origin. Preferably, but not necessarily, cancer cells excised from the patient to whom the complex is to be administered are used (e.g., the cancer cells are from one or more different individuals). May be).

[0039] Cancer and pre-neoplastic cells can be identified by any method known in the art. For example, cancer cells may have morphological, enzymatic assays, proliferation assays, cytogenetic features, DNA mapping, DNA sequences, the presence of oncogenic viruses, or a history of exposure to mutagens or oncogenic agents, It can be identified by diagnostic imaging and others. Cancer and pre-neoplastic cells can be isolated by any method known in the art. For example, cancer cells can be obtained by surgery, an endoscope, or other biopsy technique. If any clearly distinguishable characteristics are known for the cancer cell, for example, but not limited to, affinity chromatography,
Cancer cells are obtained or obtained by any biochemical or immunological method known in the art, such as and fluorescence-activated cell sorting (e.g., using a fluorescently labeled antibody to an antigen expressed on the cancer cells). It can also be purified.

There is no requirement that the population of cancer cells used to generate the cancer cDNA library must be clone-derived or homogeneous or purified. The cancerous tissue, cancer cells, or cell lines may be obtained from a single individual or pooled from several individuals. As long as at least one or more antigenic determinants on the target cancer cells are present on the cells used to create the cancer cDNA library,
It is not essential to use cells that are ultimately targeted in vivo (eg, cells from a tumor of an individual intended to be the recipient of this treatment). Furthermore, an immunogenic composition against a primary cancer can be prepared using cells derived from distant metastases. The mixture of cells that can be used has been defined such that the majority of the cells in the mixture are cancer cells and have at least one antigenic determinant in common with the target cancer cells. In certain embodiments, the cancer cells used to construct the cDNA library have been purified.

Non-covalent hsp-peptide conjugates derived directly from a cancer can be used to elicit a specific immune response against the same cancer in a patient, and thus for the prevention and treatment of that cancer. Useful. For example, PCT published WO 96/10411 and WO 97/1
See 0001. The present invention provides a method for preparing an immunogenic composition comprising a non-covalent complex of hsp with an antigenic protein or peptide of a cancer. Accordingly, such non-covalent conjugates are useful for preventing and treating a target cancer in a patient. An immunogenic composition prepared by the claimed method can enhance the immune capacity of an individual and elicit specific immunity against both pre-neoplastic cells and tumor cells. Such immunogenic compositions can also prevent the development of tumors and inhibit the growth and progression of tumor cells. The immunogenic composition elicits an inflammatory response at the tumor site, ultimately causing a reduction in tumor burden in the treated cancer patient. This immunogenic composition can be administered autologously to the individual from whom the cancerous tissue was obtained, or administered to individuals at increased risk of developing cancer due to family history or environmental risk factors. Can be.

The antigenic peptide of the present invention is bound to hsp in cancer cells or in recombinant host cells expressing cancer cDNA. Such an antigenic peptide can be a fragment of an antigenic peptide that is expressed in cancer cells, for example, a fragment of a tumor-specific antigen, or a fragment of a tumor-associated antigen. Such an antigenic peptide is a cloned cancer cDNA
Produced in cancer cDNA host cells expressing However, it is not necessary to isolate or characterize these antigens before using the method of the present invention, and it is not necessary to know what those antigens are. Further, the cancer cDNA expressed by genetic modification in the host cell preferably, but need not, contain the full length coding sequence.

Heat shock proteins (herein referred to as stress proteins) useful for the treatment and prevention of cancer can be selected from cellular proteins that meet any one of the following criteria. It is a protein that increases its intracellular concentration when cells are exposed to stressful stimuli, has the ability to bind other proteins or peptides, and binds the bound protein or peptide to adenosine triphosphate ( (ATP) in the presence or at a low pH; or a protein that exhibits at least 35% homology with a cellular protein having any of the above properties. Hsp in the complex prepared in the present invention is hsp70, hs
Examples include, but are not limited to, p90, gp96, protein disulfide isomerase alone or in combination. Preferably, the hsp is a human hsp. Preferred as a complex are those in which human hsp60, hsp70, or hsp90, and protein disulfide isomerase are non-covalently bound to a human protein antigen. In certain embodiments, the complex comprises an hsp called gp96, wherein the hsp
sp is present in the endoplasmic reticulum of eukaryotic cells and closely related to cytoplasmic hsp90.

[0044] Three major hsp families, hsp60, hsp70, and hsp90, have been identified to date. Also included are protein disulfide isomerase (PDI), and other proteins containing a thioredoxin-like domain in the endoplasmic reticulum, such as but not limited to ERp72 and ERp61. It is contemplated that hsp-peptide conjugates, including members of all of these families, including but not limited to PDI-peptide conjugates, can be prepared by practice of the present invention.

The hsp60, hsp70, hsp90 family, and protein disulfide isomerase family consist of proteins closely related to the stress protein whose sequence has, for example, more than 35% amino acid identity. Levels were found not to be changed by stress. Thus, the definition of stress protein, or heat shock protein, as used herein, includes at least 35% amino acid identity to members of these families whose expression levels in cells are enhanced in response to stressful stimuli. To 55%, preferably 55% to 75%, most preferably 75% to 85% of these other proteins, mutants, analogs, and variants.

In one embodiment of the invention, the hsps in the hsp-peptide complex prepared from the cancer cDNA host cell are native to the host cell, ie, non-covalent with the recombinant antigenic peptide of the cancer cell. The hsps that associate with the bond are those that occur naturally in the host cell.

In another embodiment, the hsps in the hsp-peptide complex are recombinant hs produced by a cancer cDNA host cell that has been engineered to express the recombinant hsp.
p. Such a recombinant hsp is non-covalently associated with the recombinant antigenic peptide in a host cell to form an hsp-peptide complex. Such a recombinant hsp can also be fused to a heterologous polypeptide, such as an immunoglobulin constant region, thereby facilitating purification of the non-covalent complex. A genetically engineered host cell has a sequence encoding hsp, which is operably linked to control sequences that drive expression of the hsp nucleic acid sequence in the host cell, It contains one or more copies of the nucleic acid sequence containing the sequence. Sequences encoding hsps, including cDNA and genomic DNA, can be any of such sequences. Recombinant hs produced in host cells or library cells
Preferably, p is of the same species as the animal species intended to receive the immunogenic composition. Recombinant human hsp is most preferred.

5.1. Preparation of Cancer cDNA Library [0048] Described here is a method for constructing a cancer cDNA library. Specifically stated is the preparation of complementary DNA (cDNA) from cancer cell RNA, insertion of the cDNA into a suitable cloning vector, and the transfer of the cloned cDNA into a suitable host organism for a cancer cDNA library. Introduction for growth and / or for production of the hsp-peptide complex.

Methods described in standard articles, for example, Methods in Enzymology, 1987,
Volume 154, Academic Press; Sambrook et al., 1989, Molecular Cloning-A Labora
tory Manual, 2nd Edition, Cold Spring Harbor Press, New York, USA; and
Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Ass
Routine molecular biological reactions used to construct and produce cancer cDNA libraries can be performed according to ociated and Wiley Interscience, New York, USA. The methods detailed below are for illustrative purposes and are not intended to be limiting. Various commercially available cDNA preparation and cDNA cloning systems can also be used to generate the cancer cDNA libraries of the present invention, according to the manufacturer's instructions.

5.1.1. Preparation of RNA Total ribonucleic acid (RNA) can be isolated from cancer cells by various methods known in the art, depending on the source and amount of the cells prior to cancer or tumor development. In order to construct a cDNA library that completely shows the genetic information expressed in cancer cells, it is preferable to obtain high-quality RNA with high molecular weight. In order to prepare high-quality RNA, a method that completely lyses cancer cells and rapidly inactivates nucleases is preferable.

One of the primary methods, which is not at all the only one that can be used in the present invention, is that a strong chaotropic agent, guanidine isothiocyanate, is added to a mild surfactant and 2-mercapto. Used to denature proteins and inactivate nucleases with ethanol or dithiothreitol,
Then, RNA is purified by ultracentrifugation (Chirgwin et al., 1979, Biochem. 24: 5294; Sadler
Et al., 1992, Curr. Genet. 21: 409-416). One-stage method (Chomczynski and Sacchi, 1987, Ana
l. Biochem. 162: 156-159; Chomczynski, 1989, U.S. Patent No. 4,843,155) can also be used, particularly when isolating RNA from small amounts of cellular material.

It is preferable that the total RNA isolated from the cancer cells be further purified before being converted into complementary DNA (cDNA). The majority of eukaryotic messenger RNA (mRNA) molecules have a polyadenylic acid (polyA) moiety at the 3 'end, and can be concentrated by affinity chromatography using oligo dT cellulose ( Aviv and Leder, 19
72, Proc. Natl. Acad. Sci., 69: 1408-1412). Denature the total RNA to expose the polyA tail. The poly A + RNA is then bound to the oligo dT cellulose and the remaining unbound RNA is washed away. Poly A + RNA is eluted by removing salts from the solution. This step can be repeated to further enrich the messenger RNA. A variety of oligo dT matrices can be used in different configurations, including but not limited to simple gravity columns, paramagnetic particles, and spin columns. Substituted oligo dT, for example, biotinylated oligo dT, can also be used. The amount and quality of the RNA thus obtained can be measured, for example, by a method such as formaldehyde agarose gel electrophoresis. Most preferred is the use of RNA enriched in poly A + RNA.

5.1.2. Preparation of Cancer cDNA Converting RNA to double-stranded cDNA can be performed in a number of different ways well known in the art. For example, Okayama and Berg, 1982, Mol. Cell Biol. 2: 161
-170; Gubler and Hoffman, 1983, Gene 25: 263-269; and Huse and Hansen, 1988,
See Strategies (Stratagene) 1: 1-3.

The first step in cDNA production involves oligonucleotide-primed synthesis of first strand cDNA by reverse transcriptase. For example, mRNA hybridized with an oligo dT primer can be copied into DNA by a reverse transcriptase such as AMV reverse transcriptase, MMLV reverse transcriptase, or Superscript (Kotewicz et al., 1988, Nucleic Acid Res. 16:
265-277). Instead of an oligo dT primer, a random hexamer can be used to prime synthesis from the internal site of the first strand mRNA, resulting in a shorter cDNA enriched for the 5 'end of long messenger RNA. Can be

The next step in the process involves synthesizing the second strand of the cDNA and producing the appropriate DNA termini for insertion into the cloning vector. Briefly, for example, the second strand
cDNA can be synthesized using E. coli DNA polymerase I and Klenow fragment, and using RNA-DNA as a template. RNA in the RNA-DNA hybrid
The gaps in the newly synthesized second strand cDNA can be removed with RNase H and filled in with E. coli DNA polymerase I. No. produced in this way
Fragments of the two-strand cDNA are ligated together using E. coli DNA ligase to create an adjacent second-strand cDNA.
Allow DNA to form.

After second-strand DNA synthesis, to form a perfectly paired (ie, blunt-ended) strand, the double-stranded DNA is further subjected to enzymes such as RNase H, RNase A, T4 DNA polymerase, And repair with E. coli DNA ligase or the like.

In cases where the amount of starting cellular material is very limited, all cDNAs obtained from the cancer cells are subjected to polymerase chain reaction (PCR) and ligation prior to cloning. Amplification can be performed in vitro by nucleic acid amplification methods known in the art, such as chain reaction (LCR). Oligo dT-primed first-strand cDNA, usually obtained by standard methods, was extended with an oligo dG tail by terminal transferase and a second primer containing an oligo dC segment was used with a thermostable DNA polymerase. Used to prime second strand synthesis. In this way, a population of double-stranded cDNAs is produced, each molecule of which is flanked by two oligonucleotides of known sequence. Standard PCR methods can be used with appropriate primer sets to amplify all of the cDNAs generated from the cancer cells. For example, U.S. Patent Nos. 4,683,202, 4,683,195, and 4,889,818; Gyllenstein et al., 1988, Proc.
cad.Sci. USA 85: 7652-7656; Ochman et al., 1988, Genetics 120: 621-623; Loh et al.,
1989, Science 243: 217-220; Tam et al., 1989, Nucleic Acid Res. 17: 1269; Bely
See avsky et al., 1989, Nucleic Acid Res. 17: 2919-2932. In certain embodiments of the present invention, RT-PCR can be used to generate amplified cDNA from cancer cell RNA (e.g., Demec et al., 1990, Anal.Biochem. 188: 422-426; Van Geld
er et al., 1990, Proc. Natl. Acad. Sci. 87: 1663-1667).

Provide a suitable restriction enzyme cleavage site suitable for imparting control functions to the DNA sequence, eg, a promoter, to the double-stranded cDNA, or for inserting the double-stranded DNA into the cloning site of the vector. The linker or adapter can be ligated to the end of the cDNA by techniques well known in the art (Wu et al., 1987, Methods in Enzymol. 152: 343-349). Modifications such as blunt ends can be made by digesting again after restriction enzyme digestion or by embedding single-stranded DNA ends prior to ligation. Alternatively, the desired restriction enzyme cleavage site can be introduced into the cDNA by amplifying the cDNA using PCR using primers containing the desired restriction enzyme cleavage site. Homopolymeric tailing can also be used to create suitable ends for cloning in cDNA (Eschenfeldt et al.
, 1987, Methods in Enzymol. 152: 337-342).

The linker is a synthetic double molecule with blunt ends at both ends. Linker to double-stranded cD
Prior to ligation to the NA, the cDNA is protected by an appropriate restriction enzyme-related modification system in order to protect the internal digestible sites of the cDNA from restriction digestion (required for ligation of the vector and linker). And methylated. For example, a double stranded cDNA can be methylated by E. coli methylase, ligated to an E. coli linker, and digested with EcoRI to create an EcoRI site at the end of the cDNA. The cDNA linked with the linker can be directly inserted into a cloning vector having an EcoRI site.

An adapter is a partially double-stranded, short DNA molecule that contains a blunt end that has been phosphorylated to be ligated to the end of the cDNA, and optionally one or more rare restriction enzyme cleavage sites. It has a heavy chain region and a single chain segment that forms compatible ends for ready insertion into a cloning vector that has a corresponding restriction enzyme cleavage site. If the adapter is used to modify the ends of the cDNA, the methylation and restriction enzyme digestion steps described above can be omitted.

Other well-known methods of making cDNAs with unique ends for use in direction-specific or directional cloning can also be used. This method doubles the possibility of expressing the cloned cDNA in the correct direction by using a cloning vector having a promoter at an appropriate position.

Briefly, for example, directional cloning can be performed by hybridizing mRNA with a linker-primer having a poly dT moiety and an internal methylation sensitive restriction enzyme cleavage site, such as XhoI. it can. The linker-primer methylates dCTP therein with a mixture of reverse transcriptase and nucleotides.
It is extended using the one that replaced TP. Upon completion of second strand synthesis, an adapter containing the desired restriction enzyme cleavage site, such as EcoRI, can be ligated to the double stranded cDNA, which is then treated with XhoI. Xh at the 3 'end of the cDNA
An oI site is created but the internal methylated XhoI site remains uncleaved. A cDNA having a desired site, such as EcoRI, at the 5 'end and an XhoI site at the 3' end can be cloned unidirectionally into a vector such that the 5 'end of the cDNA is always located downstream of the promoter. Can be

Alternatively, an adapter-primer containing a poly dT moiety in close proximity to a rare restriction enzyme cleavage site, such as NotI, can be used. Subsequent operations are similar to oligo-dT primed synthesis, except that the final cDNA to which the adapter (e.g., EcoRI adapter) has been added is digested with a rare restriction enzyme.
This is done using the unsubstituted nucleotides described above, resulting in one having the desired restriction enzyme cleavage site, eg, EcoRI, at one end and a rare restriction enzyme cleavage site at the other end. A cDNA with an EcoRI site at the 5 'end and a rare restriction enzyme cleavage site such as NotI at the 3' end can be unidirectionally cloned into a vector containing an EcoRI / NotI cloning site, There, the promoter can be located upstream of the EcoRI cloning site.

The linker-added or adapter-added cDNA can be passed through a size exclusion chromatography column, such as SEPHAROSE ™ CL-4B, to remove unlinked linkers or Adapters and other low molecular weight materials can be removed. Optionally, the linker- or adapter-added cDNA can be fractionated, for example, by agarose gel electrophoresis, to produce cDNA enriched in a particular size range.

A double-stranded cDNA made from cancer cell RNA, also referred to herein as a cancer cDNA, is linked to a DNA sequence having a regulatory function for expression in a suitable host cell, and / or Alternatively, they can be inserted into a cloning vector for propagation before expression in a suitable host cell, or directly into an expression vector,
Alternatively, sequences that promote insertion into the chromosome can be flanked.

The term expression construct, as used herein, refers to a polymorphism that includes a cancer cDNA sequence operably associated with one or more regulatory regions and allows for expression of the cancer cDNA in a suitable host cell. Means nucleotide. "Operably related" is expressed
The relationship is such that the cDNA sequence and the regulatory regions are linked and positioned such that transcription is achieved, and ultimately translation is achieved.

The control regions required for transcription of a cancer cDNA can be provided by the expression construct.
A translation initiation codon (ATG) can also be provided if one wishes to express a cancer cDNA fragment that does not include a cognate initiation codon. In compatible host-construct systems, cellular transcription factors such as RNA polymerase bind to the control regions of the expression construct and cause transcription of the cancer cDNA in the host organism. The exact nature of the regulatory regions required for gene expression can vary between host cells. Usually, a promoter is required that can bind RNA polymerase and operably promote the transcription of related nucleic acid sequences. Such control regions include 5 ′ non-coding sequences involved in the initiation of transcription and translation, such as TATA boxes, capping sequences, CAAT sequences, and the like. The non-coding region from the 3 'end to the coding region can contain transcription termination control sequences such as terminators and polyadenylation sites. It is not necessary that the control region and the cancer cDNA sequence be directly adjacent to each other to be "operably related". Appropriate regulatory regions for gene expression are well known in the art
(See Section 5.1.3).

[0068] Both constitutive and inducible control regions can be used for cancer cDNA expression. In cases where the optimal growth conditions for the host cell and the conditions for high level expression of the cancer cDNA are different, it may be desirable to use an inducible promoter. The use of inducible control regions is particularly desirable where some of the proteins encoded by the cancer cDNAs provide advantages or disadvantages to the growth of recombinant host cells expressing those proteins. Examples of useful control areas are given in the next section.

An expression construct comprising a cancer cDNA functionally related to a control region can be introduced directly into a suitable host cell for expression and production of the hsp-peptide complex without further cloning. it can. See, for example, US Patent No. 5,580,859. The expression construct can also include a DNA sequence that facilitates integration of the cancer cDNA into the genome of the host cell, eg, by homologous recombination. In this case, it is not necessary to use an expression vector containing an origin of replication suitable for a suitable host cell for propagation and expression of the cancer cDNA in the host cell.

The expression construct can also include specific oligonucleotide sequences at both ends, which can be used as primers for amplifying cancer cDNA by polymerase chain reaction (PCR). The design of primer sequences for DNA amplification and ligation of primer sequences to cancer cDNA can be performed by any method known in the art, including those using the linker and adapter described above. It is. Amplification is performed, for example, using a Perkin-Elmer Cetus thermocycler and Taq polymerase (Gen
e Amp (trade name)). Such a library of expression construct cDNAs can be amplified and maintained in vitro without the use of DNA sequences that propagate the polynucleotides in living cells. A library of expression constructs containing cancer cDNAs
It can be stored or stored by DNA amplification, and the resulting nucleic acid molecules can be split and lyophilized and / or frozen for storage. If desired, a portion of the library of expression constructs can be thawed and introduced directly into host cells for expression. Such expression constructs can be used for transient expression of a cancer cDNA in a recombinant host cell. This approach may be particularly useful where recombinant host cells are not amenable to long term culture or long term hsp-peptide complex production.

In various embodiments of the present invention, the cancer cDNA or expression construct comprising the cancer cDNA can be inserted into an expression vector for propagation and expression in a host cell as described below.

5.1.3. Host-Vector Expression System Described herein are vectors and host cell systems that can be used for cloning and expressing cancer cDNA. An expression vector is a cloning vector that can be used to maintain and express the cancer cDNA in a suitable host cell. Any cloning vector known in the art can be used to propagate cancer cDNA. Various cloning vectors can be used in the present invention, including, but not limited to, plasmids, cosmids, phages, phagemids, or modified viruses. Typically, such cloning vectors contain an origin of replication functional for propagation of the vector in a suitable host cell, one or more restriction endonuclease cleavage sites for insertion of a cancer cDNA, and one or more restriction endonuclease cleavage sites.
Contains one or more selectable markers. The cloning vector must be used with a compatible host cell, which may be prokaryotic or eukaryotic, for example, but not limited to, from bacteria, yeast, insects, mammals, and humans.

The expression constructs and vectors are introduced into host cells for the purpose of expressing cancer cDNA.
Any cell type capable of producing the stress protein and being compatible with the expression vector can be used, including those cultured in vitro or engineered. Host cells broadly include cells of unicellular organisms, such as bacteria, fungi, and yeasts, and multicellular organisms, such as insects and cells of animals, including but not limited to birds, mammals, and humans. Host cells can be obtained from normal or diseased subjects, including healthy humans and patients, from private laboratory archives, from a public culture collection such as the American Type Culture Collection, or from commercial suppliers.

Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Host cells can be selected that modify and process the expressed gene product in the same specific manner as cancer cells. Modification (eg, glycosylation) and processing (eg, cleavage) of the protein encoded by such cancer cDNAs may increase the antigenicity of the protein. In certain embodiments, normal cells obtained from the same type of tissue as the tissue that has developed cancer can be used. The type of host cell used in the present invention has been used for the expression of heterologous genes, and is preferably one that has been characterized as fully as possible and has been developed for large-scale production processes. In addition, the type of host cell is preferably non-adherent to the surface of a cell culture container such as plastic to facilitate cell recovery. In certain embodiments, the host cell is a cancer DN
It is of the same species as the patient who obtained the RNA to make A and / or of the same species as the patient to which the hsp-peptide complex is subsequently administered.

E. coli-based vectors are the most common and versatile system for high-level expression of foreign proteins (Makrides, 1996, Microbiol. Rev. 60: 512-538).
. Examples of control regions that can be used for expression in E. coli include lac, t
rp, lpp, phoA, recA, tac, T3, T7, and λP _L (Makrides, 1996, Microbiol.
Rev. 60: 512-538), but is not limited thereto. Examples of prokaryotic expression vectors include, but are not limited to, λgt vector series such as λgt11 (Huynh et al., 1984 "DNA Cloning Techniques" Volume 1: A Practical Approach (D
Glover, pp.49-78, IRL Press, Oxford) and pET vector series (Studie
r et al., 1990, Methods Enzymol 185: 60-89). However, a potential disadvantage of prokaryotic host-vector systems is that they cannot do much of the post-translational processing in mammalian cells. Thus, eukaryotic host-vector systems are preferred, mammalian host-vector systems are more preferred, and human host-vector systems are most preferred.

A variety of control regions can be used to express cancer cDNAs in mammalian host cells, including the early and late promoters of SV40, cytomegalovirus (C
MV) immediate early promoter, and Rous sarcoma virus long terminal repeat (RSV-LTR)
A promoter or the like can be used. Inducible promoters that are probably useful in mammalian cells include, but are not limited to, the metallothionein II gene, the mouse mammary tumor virus glucocorticoid-responsive long terminal repeat (MMTV-LTR), the β-interferon gene, and the hsp70 gene (Williams et al., 1989, Cancer R
es. 49: 2735-42; Taylor et al., 1990, Mol. Cell Biol. 10: 165-175). It may be advantageous to use a heat shock or stress promoter to drive expression of the cancer cDNA in the recombinant host cell. In this case, by exposing the recombinant host cell to an appropriate stress such as high temperature, the protein of the cancer cell can be expressed in the same manner as the heat shock protein or the stress protein of the recombinant host cell.

The expression efficiency of the cancer cDNA in the host cell may be determined by appropriate transcriptional enhancement elements such as those found in SV40 virus, hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, β-actin, etc. (Bittner et al., 1987, Methods in Enzymol
153: 516-544; see Gorman, 1990, Curr. Op. In Biotechnol. 1: 36-47.)
.

[0078] The expression vector can also include sequences capable of maintaining and replicating the vector in two or more types of host cells, or sequences for integrating the vector into the host chromosome. Such sequences include, but are not limited to, an origin of replication, an autonomously replicating sequence (ARS), centromere DNA, and telomeric DNA. It would also be advantageous to use a shuttle vector that can replicate and maintain in at least two types of host cells.

In addition, the expression vector can include a selectable or screenable marker gene to initially isolate, identify, or track host cells containing the cancer cDNA. For long-term, high-yield production of hsp-peptide complexes, stable expression in mammalian cells is preferred. A number of selection systems can be used for mammalian cells, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski). , 1962, Proc. Natl. Acad. Sci.
USA 48: 2026), and adenine phosphoribosyltransferase (Lowy et al., 198
0, Cell 22: 817) gene in tk ⁻ , hgprt ⁻ , or aprt ⁻ cells, respectively. Also, dihydrofolate reductase (dhfr) (which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3567;
Natl. Acad. Sci. USA 78: 1527)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad.
Sci. USA 78: 2072)); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. M.
ol. Biol. 150: 1)); and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 1).
Antimetabolite resistance to 47)) can be used as a basis for selection. Other selectable markers such as, but not limited to, histidinol and Zeos
in (trade name) can also be used.

A number of viral-based expression systems can also be used with mammalian cells to generate a cancer cDNA library. Vectors using a DNA virus backbone include Simian virus 40 (SV40) (Hamer et al., 1979, Cell 17: 725), adenovirus (Van Doren
Et al., 1984, Mol. Cell Biol. 4: 1653), adeno-associated virus (McLaughlin et al., 1988).
J. Virol. 62: 1963), and bovine papillomavirus (Zinn et al., 1982, Proc. N.
Atl. Acad. Sci. USA 79: 4897). When an adenovirus is used as an expression vector, the donor DNA sequence can be ligated to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, the E1 or E3 region) results in a recombinant virus that is viable in the infected host and can express heterologous products. (For example, Logan and Shen
Natl. Acad. Sci. USA 81: 3655-3659).

Alternatively, the vaccinia 7.5K promoter can be used. (E.g., Macket et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al.,
1984, J. Virol. 49: 857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. US
A 79: 4927-4931). When using human host cells, EB virus (EBV)
Vectors based on origin (OriP) and EBV nuclear antigen 1 (EBNA-1; trans-acting replication factor) can be used. Such vectors are used with a wide range of human host cells, for example, EBO-pCD (Spickofsky et al., 1990, DNA Prot. Eng. Tech. 2: 14-18); pDR2 and λDR2 (available from Clontech Laboratories). be able to. The expression vector pDR2 has an EBV origin that, when activated with EBNA-1, gives the vector stable episomal maintenance. When pDR2 is transfected into a cell line expressing EBNA-1, a very high transfection efficiency of 10 ^-1 can be obtained. High efficiency transfection can be achieved by first transfecting the host cell with an expression construct that produces EBNA-1.

[0082] Cancer cDNA libraries can also be generated using retrovirus-based expression cloning systems. Remove most of the viral gene sequence to remove cancer cDNA
A retrovirus, such as Moloney murine leukemia virus, can be used since it can replace lost viral functions in trans. Compared to transfection, retroviruses can efficiently infect and transfer genes to a wide range of cell types, such as primary hematopoietic cells.
Further, the range of hosts infected by the retroviral vector can be varied by the choice of the envelope used for packaging the vector.

For example, a retroviral vector contains a 5 ′ LTR, a 3 ′ LTR, a packaging signal,
Consists of a bacterial origin of replication and a selectable marker. The cancer cDNA is inserted at a position between the 5'LTR and 3'LTR such that transcription from the 5'LTR promoter results in transcription of the cloned cancer cDNA. The 5 'LTR includes, in this order, a promoter, including but not limited to the LTR promoter, an R region, a U5 region, and a primer binding site. The nucleotide sequences of these LTR elements are well known in the art. A heterologous promoter as well as multiple drug selectable markers can be included in the expression vector to facilitate selection of infected cells. McLauchlin et al., 1990, Pro
g. Nucleic Acid Res. and Molec. Biol. 38: 91-135; Morgenstern et al., 1990, Nu.
cleic Acid Res. 18: 3587-3596; Choulika et al., 1996, J. Virol. 70: 1792-1798.

[0084] Other useful eukaryotic host-vector systems include yeast and insect systems. Numerous vectors containing promoters that are constitutive or inducible in yeast, Saccharomyces cerevisiae (baker yeast), Schizosaccha
It can be used with romyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeast). As a review, Current Proto
cols in Molecular Biology, Vol. 2, 1988, Ausubel et al., Greene Publish. Ass
oc. & Wiley Interscience, Chapter 13; Grant et al., 1987, Expression and Secretio
n Vectors for Yeast, Methods in Enzymology, edited by Wu & Grossman, 1987, Acad.
Press, New York, USA, Volume 153, pp. 516-544; Glover, 1986, DNA Cloni
ng, Volume II, IRL Press, Washington DC, USA, Chapter 3; and Bitter, 1987, He
terologous Gene Expression in Yeast, Methods in Enzymology, Berger & Kim
mel, Acad. Press, New York, USA, Vol. 152, pp. 673-684; and The M
olecular Biology of the Yeast Saccharomyces, 1982, edited by Strathern et al., Cold S
See pring Harbor Press, Volumes I and II.

In an insect system, one of the baculoviruses, Autographa californica
Nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing cancer cDNA in Spodoptera frugiperda cells. Cancer cDNA sequences can be cloned into non-essential regions of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter). These recombinant viruses were then used to infect host cells, and the DN inserted into the host cells was used.
A will be expressed. (E.g. Smith et al., 1983, J. Virol. 46: 584; Smith,
See U.S. Patent No. 4,215,051. Any of the cloning and expression vectors described herein can be synthesized and assembled from known DNA sequences by techniques well known in the art. The control regions and enhancer elements can be used from various sources, both natural and synthetic. Some of the vectors and host cells are commercially available. Useful vectors include, but are not limited to, pCDM8, and λDR2 (Current Protocols in Molecular Biology, 1988
, Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference). Preferred mammalian host cells include, for example, Chinese hamster ovary (CHO) cells from humans, monkeys, and rodents, NIH / 3T3, COS, HeLa, Daudi, 293, 293-EBNA, VERO, and the like. However, the present invention is not limited thereto (for example, Kriegler M. "Gene Transfer
and Expression: A Laboratory Manual ", Newman, USA, Freeman & Co.
See 1990).

The expression host-vector system is exemplified by λDR2 in which a λ bacteriophage-based cloning vector is linked to a mammalian expression plasmid. The advantage of this system is that a highly efficient λ in vitro packaging system can be used for E. coli.
It can be used for initial library construction in a host. Size selection is not necessary as this packaging system only allows a certain range of inserts. λ vectors are usually very easy to amplify and store. The initial library in E. coli can be amplified to produce supercoiled plasmid DNA that can be used in high efficiency transformation methods for introduction into other expression host organisms. For example, λDR2 uses loxP-mediated site-specific recombination, which cuts out an expression vector pDR2 containing a cDNA insert from a λ clone that can be recircularized to generate a plasmid. Plasmid pDR2 is
The library contains eukaryotic regulatory regions based on EB virus and selectable markers to allow efficient and direct introduction of the cDNA insert into recipient human host cells.

5.1.4. Production of cDNA Libraries Expression constructs containing cloned cancer cDNAs can be introduced into host cells by a variety of techniques known in the art, including prokaryotic cells. For bacterial transformation (Hanahan, 1985, DNA Cloning, A Practical Approach, 1: 109-136), and for eukaryotic cells calcium-mediated transfection (Wig
ler et al., 1977, Cell 11: 223-232), liposome-mediated transfection (Schaefer-Ridder et al., 1982, Science 215: 166-168), electroporation.
(Wolff et al., 1987, Proc. Natl. Acad. Sci. USA 84: 3344), and microinjection (Cappechi, 1980, Cell 22: 479-488).

For long-term, high-yield production of the hsp-peptide complex, stable expression in mammalian cells is preferred. Cell lines that stably express the hsp-peptide complex can be engineered with vectors containing selectable markers. By way of example, and not by way of limitation, after introduction of the expression construct, the engineered cells are allowed to grow for 1-2 days in a nutrient-rich medium, and then are switched to a selective medium. The selectable marker in the expression construct confers resistance to selection, allowing the cells to stably integrate the expression construct into its chromosome and grow and expand in culture to a cell line.

[0089] Recombinant host cells can be cultured under conditions of standard temperature, incubation time, optical density, and medium composition. Alternatively, the recombinant host cell is a cancer
It can be cultured under conditions that compete with the nutritional and physiological requirements of the cancer cells from which the cDNA was derived. However, conditions for maintenance and production of a cancer cDNA library will be different from those for expression of an antigenic protein or peptide. Modified culture conditions and media can also be used to increase hsp-peptide complex production. For example, host cells containing the cancer cDNA can be exposed to heat or other environmental or chemical stress prior to purification of the hsp-peptide complex. Any technique known in the art can be applied to establish optimal conditions for producing the hsp-peptide complex.

5.2. Purification of hsp-Peptide Complex The following protocol can be used to recover and purify the hsp-peptide complex from mammalian cells, such as human cells, containing the expression construct containing the cancer cDNA. 5.2.1. Preparation and purification of hsp70-peptide conjugate Purification of the hsp70-peptide conjugate has been described previously. For example, Udono et al., 1993
, J. Exp. Med. 178: 1391-1396. The operations that may be used, given by way of example and not limitation, are as follows.

First, the recombinant host cells were prepared in 5 mM sodium phosphate buffer, pH 7, 150 mM N
aCl, 2 mM CaCl ₂ , 2 mM MgCl ₂ and 1 mM phenylmethylsulfonyl fluoride (P
MSF) in 3 volumes of 1 × lysis buffer. The pellet is then sonicated on ice until> 99% of the cells are lysed (as determined by microscopy). As an alternative to sonication, the cells may be lysed by mechanical shearing, in which approach the cells are typically resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF and incubated on ice for 20 minutes, Then homogenize in a Downs homogenizer until> 95% of the cells are lysed.

[0092] The lysate is then centrifuged at 1,000 g for 10 minutes to remove non-disrupted cells, nuclei and other cell debris. The resulting supernatant was centrifuged again at 100,000 g for 90 minutes, the supernatant was collected, and the Con equilibrated with phosphate buffered saline (PBS) containing 2 mM Ca ²⁺ and 2 mM Mg ^2+. Mix with A SEPHAROSE ^™ . If cells are lysed by mechanical shearing, dilute the supernatant with an equal volume of 2 × lysis buffer before mixing with Con A SEPHAROSE ^™ . The supernatant is then bound to Con A SEPHAROSE ^™ at 4 ° C. for 2-3 hours. Recover unbound material, 10 mM Tris-acetate pH 7.5, 0.1 mM
Dialyze EDTA, 10 mM NaCl, 1 mM PMSF for 36 hours (3 times, 100 volumes each time). The dialysate is then centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Next, the obtained supernatant was collected and 20 mM Tris-acetate pH 7.5, 20 mM N
Mono Q FPL equilibrated in aCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol
Add to ^CTM ion exchange chromatography column. Next, set the column to 20 mM to 500 mM.
The eluted fraction was developed with a NaCl gradient, and the eluted fraction was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a suitable anti-hsp70 antibody (for example, Stres
(from sGen clone N27F3-4) by immunoblotting.

[0093] Fractions strongly immunoreactive with the anti-hsp70 antibody are pooled and the hsp70-peptide complex is precipitated with ammonium sulfate, especially 50% to 70% ammonium sulfate. The resulting precipitate was then collected by centrifugation at 17,000 rpm (SS34 Sorvall rotor) and 70%
Wash with ammonium sulfate. The washed precipitate is then solubilized and residual ammonium sulfate is removed by gel filtration on a SEPHADEX ^™ G25 column (Pharmacia). To achieve a high degree of purification (eg, greater than 80% by weight), the hsp70 preparation thus obtained can be purified again with a Mono Q FPCL column as described above. Using this method, the hsp70-peptide complex can be purified to apparent homogeneity.
Typically, 1 mg of hsp70-peptide conjugate can be purified from 1 g of cells or tissue.

An alternative method for purifying the hsp-70 peptide complex from a cancer cDNA host cell is as follows. : Homogenize a 10 ml cell pellet of the recombinant host cells by Dounce homogenization in 40 ml of hypotonic buffer (10 mM NaHCO ₃ , 0.5 mM PMSF, pH 7.1). Subsequently, the lysate is centrifuged at 100,000 × g for 90 minutes. The supernatant was collected, PD-10 column ^{(SEPHADEX TM G-25, Pharmacia} Biotech, Piscataway, NJ) by passing through a, the buffer Buffer D (20 mM Tris-acetate, 20 mM NaCl, 15 mM B- mercaptoethanol, 3 mM Replace with MgCl ₂ , 0.5 mM PMSF, pH 7.5). The sample was applied to an ADP-agarose column (Sigma Chemical Co., St.
(Louis, MO) (5 ml) directly. The column is compatible with the Bradford protein assay (Bio
Rad, Richmond, California) until the protein can no longer be detected.
Wash with buffer D containing 0.5M NaCl, then with buffer D alone. Finally, the column was buffered with 3 mM ADP (Sigma Chemical Co, St. Louis MO).
Incubate for 30 minutes at room temperature and then elute with the same buffer (25 ml). The buffer of the eluate is exchanged again for the FPLC ^™ ion exchange chromatography buffer (20 mM Na phosphate, 20 mM NaCl, pH 7.0) using a PD-10 column. The protein in the eluate is then separated on an FPLC ^™ ion exchange chromatography system (Mono Q, Pharmacia) and eluted with a 20-60 mM NaCl gradient. 10ml
The amount of hsp70 obtained from the cell pellet is between 500 μg and 1 mg.

5.2.2. Preparation and Purification of hsp90-Peptide Complexes The procedures that can be used, given by way of example and not limitation, are as follows.

First, a recombinant host cell was prepared using 5 mM sodium phosphate buffer (pH 7), 150 mM NaC
l, ₂ mM CaCl ₂ , 2 mM MgCl ₂ and 1 mM phenylmethylsulfonyl fluoride (PMS
Suspend in 3 volumes of 1 × lysis buffer consisting of F). The pellet is then sonicated on ice until> 99% of the cells are lysed (as determined by microscopy). As an alternative to sonication, cells may be lysed by mechanical shearing, in which approach the cells are typically resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF,
Incubate on ice for 20 minutes, then homogenize in a Dounce homogenizer until> 95% of the cells are lysed.

The lysate is then centrifuged at 1,000 g for 10 minutes to remove non-disrupted cells, nuclei and other cell debris. The resulting supernatant is centrifuged again at 100,000 g for 90 minutes, the supernatant is collected and then mixed with Con A SEPHAROSE ^™ equilibrated with PBS containing 2 mM Ca ²⁺ and 2 mM Mg ²⁺ . If cells are lysed by mechanical shearing, dilute the supernatant with an equal volume of 2 × lysis buffer before mixing with Con A SEPHAROSE ^™ .
The supernatant is then bound to Con A SEPHAROSE ^™ at 4 ° C. for 2-3 hours. Recover unbound material, 10 mM Tris-acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM
Dialyze against PMSF for 36 hours (3 times, 100 volumes each time). Next, dialysate was added to 17,
Centrifuge at 000 rpm (Sorvall SS34 rotor) for 20 minutes. The resulting supernatant is then collected and applied to a Mono Q FPLC ^™ ion exchange chromatography column equilibrated with lysis buffer. The protein is then eluted with a salt gradient from 200 mM to 600 mM NaCl.

Next, the eluted fraction was fractionated by SDS-PAGE, and an anti-hsp90 antibody, for example, 3G3 (Affi
The fractions containing the hsp90-peptide complex are identified by immunoblotting, using Biochemistry (Nature Bioreagents). Using this procedure, an apparently homogeneous preparation of the hsp90-peptide complex can be obtained. Typically, the hsp 90-peptide complex 150
200200 μg can be purified from 1 g of cells or tissue.

5.2.3. Preparation and Purification of gp96-Peptide Complexes The procedures that can be used, given by way of example and not limitation, are as follows.

The pellet of recombinant host cells was washed with 30 mM sodium bicarbonate buffer (pH 7.5) and
Resuspend in 3 volumes of buffer consisting of mM PMSF and swell cells on ice for 20 minutes. The cell pellet is then homogenized in a dounce homogenizer on ice until> 95% of the cells are lysed (the appropriate gap in the homogenizer will vary for each cell type).

The lysate is centrifuged at 1,000 g for 10 minutes to remove unbroken cells, nuclei and other debris. The supernatant from this centrifugation step is then centrifuged again at 100,000 g for 90 minutes. The gp96-peptide complex can be purified from the pellet or supernatant after centrifugation at 100,000 g.

When purifying from the supernatant, the supernatant is diluted with an equal volume of 2 × lysis buffer and then at 4 ° C. the supernatant is diluted with PBS containing 2 mM Ca ²⁺ and 2 mM Mg ²⁺ . Mix with equilibrated Con A SEPHAROSE ^™ for 2-3 hours. Thereafter, the slurry is loaded onto a column and washed with 1 × lysis buffer until OD ₂₈₀ drops to baseline. Next, the column was washed with 1/3 of the column bed volume with 10% α-methyl mannoside (α-MM) dissolved in PBS containing 2 mM Ca ²⁺ and 2 mM Mg ²⁺ , and one piece of parafilm Seal the column with and incubate at 37 ° C for 15 minutes. Next, the column is cooled to room temperature and parafilm is removed from the bottom of the column. Add 5 column volumes of α-MM buffer to the column and analyze the eluate by SDS-PAGE. Typically, the purity of the resulting material is between about 60% and 95%, depending on the cell type and the tissue to lysis buffer ratio used. This sample is then added to a Mono Q FPLC ^™ ion exchange chromatography column (Pharmacia) equilibrated with a buffer (pH 7) containing 5 mM sodium phosphate. Then, 0-1M NaCl
When the protein is eluted from the column using a gradient, the gp96 fraction elutes in the range of 400 mM to 550 mM NaCl.

However, the procedure may be modified by utilizing two additional steps, alone or in combination, such that an apparently uniform gp96 peptide complex is consistently formed. In one optional step, the ammonium sulfate precipitation prior to the Con A purification step and, on the other hand any step, the DEAE-SEPHAROSE ^TM purified prior to and Mono Q FPLC ^TM ion exchange chromatography step after the Con A purification step .

In a first optional step, ammonium sulfate is added to the supernatant obtained from the 100,000 g centrifugation step so that the final concentration of ammonium sulfate is 50%. In a beaker placed in a tray with ice water, slowly add ammonium sulfate while gently stirring the solution. Approximately 1 solution at 4 ° C
Stir for 1/2 hours to 12 hours and centrifuge the resulting solution at 6,000 rpm (using a Sorvall SS34 rotor). Take out the supernatant obtained in this step, add aluminum sulfate to make 70% aluminum sulfate saturated solution, and centrifuge at 6,000 rpm (Sorvall SS34
With rotor). The pellet obtained in this step is collected, suspended in PBS containing 70% ammonium sulfate, and the pellet is washed. The mixture is centrifuged at 6,000 rpm (using a Sorvall SS34 rotor) and the pellet is dissolved in PBS containing 2 mM Ca ²⁺ and Mg ²⁺ . Centrifuge briefly at 15,000 rpm to remove undissolved material (using a Sorvall SS34 rotor). This solution is then mixed with Con A SEPHAROSE ^™ and the procedure described above is repeated.

In a second optional step, the gp96-containing fractions eluted from the Con A column were pooled,
The buffer is exchanged for 5 mM sodium phosphate buffer (pH 7, 300 mM NaCl) by dialysis or preferably by buffer exchange using a SEPHADEX ^™ G25 column. After replacing the buffer, the solution was washed with 5 mM sodium phosphate buffer (pH 7, 3
Mix with DEAE-SEPHAROSE ^™ previously equilibrated with (00 mM NaCl). The protein solution and beads are mixed gently for 1 hour and poured onto the column. Next, 5 mM sodium phosphate buffer (until the absorbance at 280 nm drops to baseline).
The column is washed with (pH 7, 300 mM NaCl). Next, the bound protein is eluted from the column using 5 volumes of 5 mM sodium phosphate buffer (pH 7, 700 mM NaCl). The protein-containing fractions were pooled and 5 mM sodium phosphate buffer (pH
Dilute in step 7) to reduce the salt concentration to 175 mM. Then, the resulting material subjected to Mono Q FPLC ^TM ion exchange chromatography column equilibrated with 5mM sodium phosphate buffer (pH7) (Pharmacia), as described above, Mono Q FPLC ^TM ion exchange chromatography Elute the protein bound to the column (Pharmacia).

However, one of ordinary skill in the art would be able to evaluate the benefits of incorporating the second optional step into a purification protocol by routine experimentation. Further, it is believed that the benefits of adding each optional step will depend on the source of the starting materials.

When isolating the gp96 fraction from a 100,000 g pellet, 5 volumes of PBS containing either 1% sodium deoxycholate or 1% octylglucopyranoside (but Mg ²⁺ and Ca ²⁺ (Not included) and incubate on ice for 1 hour. This suspension is centrifuged at 20,000 g for 30 minutes, and the resulting supernatant is dialyzed against PBS (again, not containing Mg2 ⁺ and Ca2 ⁺ ), which is replaced several times. , Remove the surfactant. The dialysate is centrifuged at 100,000 g for 90 minutes, the supernatant is collected, and calcium and magnesium are added to the supernatant to a final concentration of 2 mM each. The sample is then purified, as described above, by either the unmodified or modified methods, and the gp96-peptide is isolated from 100,000 g of supernatant.

Using this procedure, the gp96-peptide can be purified to apparent homogeneity. About 10-20 μg of gp96 can be isolated from 1 g of cells or tissue.

5.2.4. Preparation and Purification of PDI-Peptide Conjugates The procedures that can be used, given by way of example and not limitation, are as follows.

The pellet of the recombinant host cells was mixed with 30 mM sodium bicarbonate buffer (pH 7.5) and 1
Resuspend in 3 volumes of buffer consisting of mM PMSF and swell cells on ice for 20 minutes. The cell pellet is then homogenized in a dounce homogenizer on ice until> 95% of the cells are lysed (the appropriate gap in the homogenizer will vary for each cell type).

The lysate is centrifuged at 1,000 g for 10 minutes to remove unbroken cells, nuclei and other debris. The supernatant from this centrifugation step is then centrifuged again at 100,000 g for 90 minutes. Ammonium sulfate is added to the supernatant so that the final concentration of ammonium sulfate is 80%. In a beaker placed in a tray with ice water, slowly add ammonium sulfate while gently stirring the solution. The solution was stirred at 4 ° C. for about 1/2 hour to 12 hours, and the resulting solution was
Centrifuge at 0 rpm (using a Sorvall SS34 rotor). The resulting pellet is collected and dissolved in PBS containing 2 mM Ca ²⁺ and 2 mM Mg ²⁺ (2 × lysis buffer). In 4 ° C., the solution, and Con A SEPHAROSE ^TM equilibrated with PBS containing 2 mM Ca ²⁺ and 2 mM Mg ^2+, and mixed for 2-3 hours, to further Hama charging the slurry into the column, OD ₂₈₀ Wash with 1 × lysis buffer until drops to baseline. The effluent is collected and the buffer is exchanged with 0.025 mM sodium citrate (pH 5.1) through a PD-10 column (Pharmacia). The solution is loaded onto a CM-Sephadex 50 (Pharmacia) cation exchange chromatography column and the effluent is collected. PD-10 column (
(Pharmacia) after buffer exchange to 0.02 M sodium phosphate and 0.1 M NaCl (pH 6.0), and then to DEAE anion exchange chromatography column washed with 0.15 M NaCl, 0.02 M sodium phosphate buffer (pH 6.0). , Pass the solution through. The column is eluted with a 0.15M to 0.5M sodium chloride gradient.

Using this procedure, the PDI-peptide conjugate can be purified to an apparent homogeneity. About 3-7 μg of protein disulfide isomerase can be isolated from 1 g of cells or tissue.

5.3. Determination of the Immunogenicity of the Stress Protein-Peptide Complex In any manner , the purified stress protein-peptide complex can be purified using a mixed lymphocyte targeted culture assay (MLTC) well known in the art. Can be assayed for immunogenicity.

As an example, the following method can be used, but the present invention is not limited to this. Briefly, mice are injected subcutaneously with the stress protein-peptide complex of interest. Other mice are injected with either other stress protein-peptide conjugates derived from normal non-recombinant cells, or with all infected cells as a positive control in this assay. Mice are injected twice every 7-10 days. Ten days after the last immunization, the spleen is removed and lymphocytes are collected. The resulting lymphocytes can then be restimulated in vitro by adding dead cells that have expressed the complex of interest. For example, 8 × 10 ⁶ immune spleen cells were treated with mitomycin C in 3 ml of RPMI medium containing 10% fetal calf serum or gamma-irradiated (5-10,000 rads) and 4 × 10 ⁴ infected cells (or (A cell transfected with an appropriate gene). In some cases, as a source of T cell growth factor, the culture medium may contain 33% secondary mixed lymphocyte culture supernatant (Glasebrook et al., 1980, J. Exp. Med. 1
51: 876). To test the primary cytotoxic T cell response after immunization,
Spleen cells may be cultured without stimulation. In some experiments, the spleen cells of immunized mice can be restimulated with cells of different antigenicity to determine the specificity of the cytotoxic T cell response.

After 6 days, cultures are tested for cytotoxicity in a 4 hour ⁵¹ Cr-release assay (
Palladino et al., 1987, Cancer Res. 47: 5074-5079 and Blachere et al., 1993, J.
Immunotherapy 14: 352-356). In this assay, different effector-to-target (E: T) ratios are obtained when mixed lymphocyte cultures are added to a target cell suspension (typically 1: 1 to 40: 1). 1 × 10 ⁶ target cells in 200 mCi of ⁵¹ Cr / ml containing medium
Target cells are pre-labeled by incubating at 37 ° C. for 1 hour. After labeling, the cells are washed three times. Each assay point (E: T ratio) was performed in triplicate, incorporating appropriate controls and spontaneous ⁵¹ Cr release (assay without added lymphocytes) and 100% release (assay without lymphocytes).
(Cells lysed with detergent). After incubating the cell mixture for 4 hours, the cells are pelleted by centrifugation at 200 xg for 5 minutes. ⁵¹ C released into the supernatant
Measure the amount with a gamma counter. The% cytotoxicity is measured as cpm determined by the following formula: (test sample-spontaneous release cpm) ÷ (total surfactant release cpm-spontaneous release cpm).

To block the MHC class I cascade, K-44 hybridoma cells (anti-MHC
Add the concentrated hybridoma supernatant from a Class I hybridoma) to the test sample to a final concentration of 12.5%.

5.4. Formulations A non-covalent complex of hsp and an oncoprotein or peptide of the invention, prepared by the method of the invention as claimed, may be formulated to treat or prevent cancer. It can be a pharmaceutical preparation for administration to mammals for the purpose. Drug solubility and absorption site are factors to consider in choosing a route of administration of a therapeutic agent. The hsp-antigenic molecule conjugates of the present invention can be administered by any desired route of administration, including but not limited to subcutaneous, intravenous or intramuscular, Or mucosa is preferred. The advantages of intradermal or mucosal administration include lower doses and rapid absorption, respectively. Mucosal routes of administration include, but are not limited to, oral, rectal and nasal administration. Preparations intended for mucosal administration are suitable for the various formulations described below. The route of administration may vary during the course of treatment. Suitable dosages, routes of administration and therapeutic regimens are described herein and in PCT International Patent Applications published as WO 96/10411 and WO 97/10001, which are hereby incorporated by reference in their entirety. Incorporate.

In a preferred embodiment, the amount of hsp70- and / or gp96-peptide conjugate administered to a human is in the range of about 10-600 μg, preferably 10-100 μg, most preferably about 25-100 μg.
μg, administered once a week for about 4 to 6 weeks, and administered to the skin or mucous membrane by changing the administration site sequentially. The preferred amount of hsp90-peptide molecule complex is 50-5,000
It is in the range of μg, preferably 100 μg.

A composition comprising a non-covalent complex, formulated in a compatible pharmaceutical carrier, can be prepared, packaged, and labeled for treatment of a tumor, as follows.
Examples of tumors include human sarcomas and carcinomas, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, lubricity Melanoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland Cancer, papillary carcinoma, papillary adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, bilumus tumor, cervix Cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma,
Medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia For example,
Acute lymphocytic and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myeloid (granular cell) leukemia and chronic lymphocytic leukemia) ), Polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, and heavy chain disease.

If the conjugate is water soluble, the conjugate can be formulated in a suitable buffer, eg, phosphate buffered saline or other physiologically compatible solution. Alternatively, if the obtained complex is hardly soluble in an aqueous solvent, the complex is
It may be formulated with a nonionic surfactant such as n or polyethylene glycol. The non-covalent complex and the physiologically acceptable solvate can then be formulated for inhalation or insufflation (through the mouth or nose) or for oral, buccal, parenteral, rectal administration. Alternatively, in the case of tumors, they can be injected directly into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquid form, for example, a solution, syrup or suspension, or for reconstitution with water or other suitable vehicle before use. It may be a drug product. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives. Such additives include suspending agents (eg, sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifiers (eg, lecithin or acacia), non-aqueous vehicles (eg, almond oil, oily esters or fractionated vegetable oils) ) And preservatives (e.g., methyl p-hydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid). Pharmaceutical compositions can be, for example, in the form of tablets or capsules, and prepared by conventional methods using pharmaceutically acceptable excipients. Such excipients include binders (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (eg, lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants ( For example, magnesium stearate, talc or silica), disintegrating agents (eg, potato starch or sodium starch glycolate) or wetting agents (eg, sodium lauryl sulfonate). Tablets may be coated by methods well known in the art.

Preparations for oral administration may be suitably formulated to give controlled release of the complex. Such compositions can be formulated in the form of tablets or drops by conventional methods.

For administration by inhalation, the complexes may conveniently be delivered in the form of an aerosol spray preparation from compressed packs or nebulisers using a suitable propellant. Suitable propellants include, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of compressed aerosols, the dosage unit can be determined by installing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the conjugate and a suitable powder base such as lactose or starch.

The conjugate can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile and pyrogen-free water, before use.

The complexes can also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the complexes may also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the conjugate may be formulated with a suitable polymeric or hydrophobic material (eg, as an emulsion in an acceptable oil) or an ion exchange resin, or as a slowly soluble derivative. For example, it may be formulated as a salt that dissolves slowly. Liposomes and emulsions are well-known examples of delivery vehicles or carriers for hydrophilic drugs.

The compositions may, if desired, be presented in a pack or in a dispenser device which may contain one or more unit dosage forms containing the non-covalent complex. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The present invention also provides kits for performing the treatment regimen of the present invention. Such kits comprise a therapeutically or prophylactically effective amount of a non-covalently bound hsp-peptide complex in one or more containers in a pharmaceutically acceptable form. The hsp-peptide conjugate in the vial of the kit of the invention may be a pharmaceutically acceptable solution, such as in combination with sterile saline, dextrose solution or buffer solution, or other pharmaceutically acceptable sterile liquid. It can be in the form of Alternatively, the complex may be lyophilized or dried in a desiccator. In this case, the kit may optionally be injected with a pharmaceutically acceptable solution (eg, saline, dextrose solution, etc.), preferably sterile, in a container by returning the complex to a liquid form. For the purpose of preparing a solution for use.

In another embodiment, the kit of the present invention further comprises a needle or syringe for injecting said conjugate, preferably packaged in sterile form, and / or a packaged alcohol pad. Instructions may be provided by clinician or patient
Optionally included for administration of the hsp-peptide conjugate.

5.5. Prevention and Treatment of Cancer A number of reasons why the immunotherapy provided by the non-covalent hsp-peptide conjugate prepared according to the present invention is desirable for use in cancer patients are listed. it can. First, if a cancer patient is immunosuppressed, surgery with anesthesia, followed by chemotherapy, can exacerbate immunosuppression. Thus, appropriate immunotherapy before surgery can prevent or reverse this immunosuppression. This reduces infectious complications and promotes wound healing. Second, the total tumor volume after surgery is minimal,
In this situation, immunotherapy may be most effective. The third reason is that during surgery, tumor cells are flushed into the circulatory system, and effective immunotherapy applied at this time can eliminate these cells. In certain embodiments, the prophylactic and therapeutic effects of the present invention are directed to enhancing the immunity of pre-, intra- or post-operative cancer patients, and inducing tumor-specific immunity to cancer cells. I have. In this case, the goal is cancer inhibition and the ultimate clinical goal is overall cancer regression and eradication.

A preferred method of treating or preventing cancer according to the present invention comprises isolating and isolating RNA molecules from cancer cells obtained from one or more individuals, preferably those in need of treatment. Producing a cDNA from the obtained RNA molecule. The cancer cDNA may be a cDNA molecule in the form of an expression construct, or integrated into a chromosome, by the methods described above in Section 5.1.
Manipulate as appropriate to express the cDNA in one or more preselected host cells.

A recombinant host cell containing the cDNA molecule is cultured under conditions such that the peptide encoded by the cDNA molecule is expressed by the recombinant host cell. The heat shock protein complex non-covalently linked to one or more peptides is recovered from recombinant host cells and / or preferably purified by the methods described in Section 5.2. The hsp-peptide conjugate is formulated as described in Section 5.4, depending on the route of administration, and is further administered to the individual in an autologous manner (e.g., to treat a primary cancer or its own metastases). Or to another individual in need of treating cancer of the same tissue type, or an individual at increased risk for cancer due to family history or environmental risk factors. Typical therapeutic and prophylactic uses of hsp-peptide conjugates are described in PCT publications WO 96/10411 (April 11, 1996) and WO 97/10001 (March 20, 1997).
Date).

In certain embodiments, the invention provides methods for treating or preventing cancer in an individual. The treatment or prevention of cancer in this individual desirably involves the following steps: (a) A host cell containing a cDNA molecule made from an RNA molecule of a cancer tissue obtained from one or more human individuals is transformed into the cDNA. Culturing under conditions such that the protein encoded by the molecule is expressed in the host cell; (b) recovering the heat shock protein complex non-covalently bound to one or more peptides from the host cell And optionally purifying the recovered complex; and (c) administering to the subject the recovered or purified complex.

In another specific embodiment, the invention provides a method of treating an individual having cancer comprising the steps of: (a) an RNA molecule derived from a cancer cell obtained from the individual. (B) producing a cDNA molecule from the RNA molecule; (c) introducing the cDNA molecule into one or more host cells in a form suitable for expression of the cDNA molecule; d) culturing a host cell containing the cDNA molecule under conditions such that the protein encoded by the cDNA molecule is expressed by the host cell; (e) non-covalently binding one or more peptides. Recovering the heat shock protein complex bound in step (a) from the host cell and optionally purifying the recovered complex; and (f) administering the recovered or purified complex to the individual.

[0135] Cancers that can be treated or prevented using the non-covalent hsp-peptide conjugates prepared by the methods of the present invention include, but are not limited to, human sarcomas, carcinomas, such as the following examples: For example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, smoothing Myoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary carcinoma, Cystic carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, bilmus tumor, cervical carcinoma, testicular carcinoma, lung carcinoma, small cell lung carcinoma, Bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymal Cysts, pineomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas, leukemias such as acute lymphoblastic leukemia and acute myeloid leukemia (Myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia)
Chronic leukemia (chronic myeloid (granular cell) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, As well as heavy chain disease. Specific examples of those cancers are described in the following sections. In certain embodiments, the cancer is metastatic. In other specific embodiments, the patient with cancer is immunosuppressed because he / she received an anti-cancer treatment (eg, chemotherapy, radiation) prior to administering the hsp-peptide molecule conjugate of the invention. . In another particular aspect, the cancer is a tumor.

The effect of immunotherapy with the hsp-peptide conjugate on the progression of a neoplastic disease can be monitored by any method known to those skilled in the art. For example, but not limited to, a) measurement of delayed hypersensitivity reactions as an assessment of cellular immunity, b) in
measurement of the activity of cytolytic T lymphocytes in vitro, c) tumor-specific antigens, for example
Measurement of carcinoembryonic antigen (CEA) levels, d) measurement of morphological changes in tumors using techniques such as computer-assisted tomography (CT) scans, e) specific cancers in high-risk individuals. Measuring changes in the level of putative biomarkers indicative of risk, and f) Measuring morphological changes in tumors using sonograms. Other techniques that can be used include scintigraphy and endoscopy.

For example, to measure the activity of cytolytic T cells in vitro, 8 × 10 ⁶ peripheral blood-derived T lymphocytes are isolated by Ficoll-Hypaque centrifugation gradient. The cells are restimulated with 4 × 10 ⁴ tumor cells treated with mitomycin C in 3 ml of RPMI medium containing 10% fetal bovine serum. In some experiments, 33% secondary mixed lymphocyte culture supernatant or IL-2 was added to the medium as a source of T cell growth factors. To measure the primary response of cytolytic T-lymphocytes after immunization, T cells are cultured without stimulatory tumor cells. In other experiments, T cells are restimulated using cells of different antigenicity. Six days later, the cultures are tested for cytotoxicity in a 4 hour ⁵¹ Cr release assay. The spontaneous ⁵¹ Cr release of the target should be at a level of less than 20%. For anti-MHC class I blocking activity, add 10-fold concentrated supernatant of W6 / 32 hybridoma to the test system to a final concentration of 12.5% (Heike, M. et al., 1994, J
Immunotherapy 15: 165-174).

[0138] An alternative to the chromium-release assay is the ELISPOT assay, which measures cytokine release by cytotoxic T cells after stimulation with a particular antigen in vitro. Cytokine release is detected by specific antibodies to specific cytokines, such as interleukin 2, tumor necrosis factor α or interferon γ (see, eg, Scheibenbogen et al., 1997, Int. J. Cancer, 71: 932-936. See). The assay is performed in a microtiter plate pre-coated with an antibody specific for the cytokine of interest, allowing the cytokine secreted by T cells to be captured. After incubating the T cells in the coated wells for 24-48 hours, the cytotoxic T cells are removed and a secondary labeled antibody that recognizes a different epitope on the cytokine is added instead. After removing unbound antibody by thorough washing, an enzyme substrate that produces a colored reaction product is added to the plate.
The number of cytokine producing cells was counted under a microscope. This method has the advantages of a short assay time and high sensitivity without requiring large amounts of cytotoxic T cells.

As an example, the immune response in a subject after immunization was measured as follows using IFN-γ ELI
It can be evaluated by the SPOT assay kit (Mabtech, Sweden). Blood samples (20 ml) obtained from patients are heparinized and peripheral blood mononuclear cells are separated from the blood samples by Ficoll (Pharmacia) gradient centrifugation. Separated blood cells can be stored at −130 ° C. as aliquots of 5 × 10 ⁶ cells. CD
8+ T cells are isolated from blood cells by standard techniques, such as using magnetic beads (Dynal). An HA-multiscreen plate (Millipore) is coated with 100 μl of a mouse anti-human IFN-γ antibody (10 μg / ml). Plate CD8 + T cells at a concentration of 8 × 10 ⁴ cells per well. Add tumor cells (5 × 10 ⁴ cells per well) or tumor cell membrane (equivalent to 5 × 10 ⁷ cells per well) and incubate at 37 ° C. for 20 hours. After incubation, the cells are removed by thorough washing with PBS / 0.05% Tween 20, and 100 μl of biotinylated capture antibody against human IFN-γ is further concentrated.
Add at 2 μg / ml. The spots are developed using standard techniques, and the spots are counted at 40 × magnification using a stereomicroscope. Each spot is a single secretion of IFN-γ
Corresponds to CD8 + T cells.

The prophylactic effect of immunotherapy using the hsp-peptide conjugates can also be estimated by measuring the level of putative biomarkers for the risk of a particular cancer. For example, in individuals at increased risk for prostate cancer,
Et al., 1992, J. Urol. 147: 841-845, and Catalona et al., 1993, JAMA 270: 948-958.
Measuring serum prostate specific antigen (PSA), or in individuals at risk for colorectal cancer, CEA by methods known in the art, , In individuals at increased risk for breast cancer,
Natl. Acad. Sci. USA 79: 3047-3051, Schneider et al., 1982, measuring 16-α-hydroxylation of estradiol. The references cited above are hereby incorporated by reference in their entirety.

[0141] 6. Example: hepatocellular carcinoma treatment hepatocellular carcinoma liver biopsy samples obtained from patients with (approximately 10 ⁶ to 10 ⁷ cells, or tissue 200Mg～1g), guanidinium isothiocyanate And gently dissolve in the presence of phenol / chloroform. After centrifuging the cell lysate,
Extract 1-2 mg of total RNA from 1 g and precipitate with isopropanol. Total poly-A ⁺ RNA is isolated from total RNA by column chromatography using commercially available pre-packaged oligo-dT cellulose spin columns (Clontech Laboratories, CA). About 50
μg of poly-A ⁺ RNA is obtained from 1 mg of total RNA.

A commercially available cDNA synthesis kit is available from the vector λDR2 (Clontech Laboratories, CA) with BamH
From poly-A ⁺ RNA with appropriate ends, unidirectionally cloned into the I / XbaI site, cD
Used to make NA molecules. This cloning vector contains an Epstein-Barr vector that provides elements for stable gene expression in the recipient human cell line.
Includes virus shuttle vector pDR2 embedded. For the purpose of expression in human cells, the 5 'end of the cDNA was replaced with the Rous sarcoma virus long terminal repeat (RSV LT
R) into the BamHI site downstream of the promoter.

The cloned cDNA was prepared in vitro using a commercially available E. coli cell extract.
And store a portion of the packaged library as lambda phage particles. The rest of the library is reconstituted by infecting E. coli AM1 host cells containing Cre recombinase. By site-specific recombination through the loxP site of this vector, the cloned cDNA is converted into the pDR2 plasmid carrying the expression construct. Expression plasmids are purified from E. coli cells and transfected into 293-EBNA cells (Invitrogen, CA), a human cell line, the neomycin resistance gene and Epstein-Estryn, which allows the expression plasmid to be stably maintained episomally. The constitutive expression of the bar virus nuclear antigen. The plasmid is maintained at about 10-20 copies per cell, and the cloned cDNA is
Expressed by the V LTR promoter.

The transfected human cells are batch- or continuous-cultured and recombinantly produced cloned cDNA. Heat shock protein-peptide complexes are purified from recombinant cells as described in Section 5.2.

Treatment with the hsp-antigen conjugate prepared as described above is initiated at any time after surgery. However, if the patient is undergoing chemotherapy, the hsp-antigen complex is usually administered at intervals of 4 weeks or more to restore the immune system. The patient's immune competence is tested by the procedure described above in Section 5.9.

A therapeutic regimen involves weekly administration of the hsp-antigen conjugate dissolved in saline or other physiologically compatible solution.

The dosage used for hsp70 or gp96 is in the range of 10-600 μg, and the preferred dose for human patients is in the range of 10-100 μg. The working dose of hsp90 is in the range of 50-5,000 μg, and the preferred dose for human patients is 50-200 μg, for example about 100 μg.

The route and site of injection will vary at each time point, eg, the first injection will be subcutaneous to the left arm, the second injection will be subcutaneous to the right arm, and the third injection will be left abdominal. Subcutaneous administration to the region, the fourth injection is subcutaneous to the right abdominal region, the fifth injection is subcutaneous to the left thigh, and the sixth injection is subcutaneous to the right thigh. , Etc. Injections at the same site are repeated after one or more injections.
In addition, the injections are split and each half of the dose is administered to different sites on the same day.

Overall, every other week for the first 4-6 injections, then every other week for two injections, then every other month the injection regimen is performed. The effects of hsp-antigen complex therapy include: a) measurement of delayed hypersensitivity to assess cellular immunity, b) cytolytic T
Measurement of the in vitro activity of cells, c) measurement of the level of tumor-specific antigens, eg carcinoembryonic antigen (CEA), d) morphology of the tumor using techniques such as computer-assisted tomography (CT) scanning E) measuring changes in the level of putative biomarkers indicative of the risk for a particular cancer in high-risk individuals;
Monitor by

With the ultimate goal of achieving tumor regression and complete eradication of cancer cells, depending on the results obtained, the treatment regimen will be reviewed to maintain and / or enhance the patient's immunological response. .

[0151] The scope of the present invention is not limited by the particular embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications have been cited herein, the disclosures of which are incorporated herein by reference in their entirety.

[Brief description of the drawings]

FIG. 1 is a flow chart illustrating an example of the method of the present invention for producing an hsp-peptide complex. Optional steps are indicated by dashed lines.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/00 A 15/09 C12N 5/00 B C12P 21/00 15/00 A (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA ( AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW F term (reference) 4B024 AA01 BA36 CA04 DA03 DA06 DA12 EA03 EA04 EA06 GA14 HA01 4B064 AG01 CA10 CA19 CC24 DA01 DA05 4B065 AA90X AA93X AA93Y AB01 BA02 CA24 MM

Claims (50)

[Claims] 1. A method for preparing a complex of a heat shock protein non-covalently bound to a peptide, comprising: (a) producing a cDNA molecule from an RNA molecule of a cancer cell or preneoplastic cell; b) ligating the cDNA molecules to an expression construct such that substantially each cDNA molecule is operably linked to at least one regulatory region that controls expression of the cDNA molecule to create a library of expression constructs. (C) introducing the library into one or more host cells; (d) subjecting the host cells containing the cDNA molecules to conditions under which the proteins encoded by the cDNA molecules are expressed by the host cells. (E) recovering a complex of heat shock protein non-covalently bound to one or more peptides from said host cell. 2. A method for preparing a complex of a heat shock protein non-covalently bound to a peptide, comprising: (a) in one or more host cells, an RNA molecule of a cancer cell or a pre-neoplastic cell; Introducing a cDNA molecule prepared from (c), provided that each cDNA molecule is operatively linked to at least one regulatory region that controls the expression of the cDNA molecule in the host cell; (b) Culturing a host cell containing the cDNA molecule under conditions in which the protein encoded by the cDNA molecule is expressed by the host cell; (c) non-covalently binding to the one or more peptides from the host cell. Recovering the bound heat shock protein complex. 3. A method for preparing a heat shock protein complex non-covalently bound to a peptide, comprising: (a) a host containing a cDNA molecule prepared from an RNA molecule of a cancer cell or a preneoplastic cell. Culturing the main cell under conditions in which the protein encoded by the cDNA molecule is expressed by the host cell; (b) the host non-covalently bound to one or more peptides from the host cell Recovering the complex of the heat shock protein of the cell. 4. A method for preparing a heat shock protein complex non-covalently bound to a peptide, comprising: (a) isolating RNA molecules from cancer cells or pre-neoplastic cells obtained from a human individual. (B) producing a cDNA molecule from the RNA molecule; (c) introducing the cDNA molecule into one or more host cells in a form suitable for expression of the cDNA molecule; culturing a host cell containing the cDNA molecule under conditions in which the protein encoded by the cDNA molecule is expressed by the host cell; (e) non-covalently binding from the host cell to one or more peptides Recovering the heat shock protein complex. 5. A method for preparing a heat shock protein complex non-covalently bound to a peptide, comprising: (a) a host comprising a cDNA molecule prepared from an RNA molecule of a cancer tissue obtained from a human individual; Culturing the cell under conditions in which the protein encoded by the cDNA molecule is expressed by the host cell; (b) removing the host cell from the host cell non-covalently bound to one or more peptides. Recovering the complex of the heat shock protein. 6. A method for preparing a complex of a heat shock protein non-covalently bound to a peptide, comprising: (a) converting a cDNA molecule prepared from an RNA molecule of a cancer cell or a pre-neoplastic cell into the cDNA molecule; Introducing the molecule into one or more host cells such that the molecule is expressed by the host cell; (b) transducing the host cell containing the cDNA molecule so that the protein encoded by the cDNA molecule is expressed by the host cell; Culturing under conditions, and (c) recovering the host cell heat shock protein complex non-covalently bound to one or more peptides from the host cell. 7. The method of claim 1, wherein the RNA molecule is isolated from cancer cells of two or more human individuals. 8. The method of claim 1, wherein said host cell is a human cell. 9. The method of claim 5, wherein said host cell is a human cell. 10. The method of claim 1, wherein said RNA molecule is total poly A ⁺ RNA. 11. The method of claim 5, wherein said RNA molecule is total poly A ⁺ RNA. 12. The method of claim 8, wherein said RNA molecule is total poly A ⁺ RNA. 13. The method according to claim 13, wherein the cancer tissue is excised from one human individual,
6. The method of claim 5, wherein said RNA molecule is total poly A ⁺ RNA and said host cell is a human host cell. 14. The method of claim 6, wherein step (c) comprises purifying the complex. 15. The method of claim 13, wherein step (b) comprises purifying said complex. 16. The method of claim 1, 2, 3, or 4, wherein said RNA molecule is obtained from a cancer cell of a tumor, leukemia or cancer cell line. 17. The method of claim 1, 2, 3, or 4, wherein said RNA molecule is obtained from a pre-neoplastic cell. 18. The method of claim 1, wherein said cancer cells are of a human cancer cell line.
5. The method according to 3 or 4. 19. The method of claim 1, wherein said RNA molecule is obtained from a metastatic cancer cell. 20. The cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma. , Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer,
Ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma , Liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, fetal carcinoma, vilmoma, cervical cancer, testis cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma , Medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia 7. The method of claim 1, 3, 5, or 6, wherein the method is selected from the group consisting of: lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, and heavy chain disease. 21. The method of claim 1, wherein said RNA molecule is a messenger RNA molecule.
5. The method according to 3 or 4. 22. The method according to claim 1, wherein the step (a) and the step (b) or the step (b) and the step (c)
2. The method of claim 1, further comprising amplifying the cDNA molecule. 23. The method of claim 4, further comprising amplifying said cDNA molecule prior to step (c). 24. The method of claim 1, further comprising, before step (c), freezing the cDNA library of step (b) and thawing the library. 25. Prior to step (c), introducing said library into an intermediate host cell, culturing said intermediate host cell to expand said library, and isolating said library from said intermediate host cell The method of claim 1, further comprising each step. 26. The expression construct, wherein the expression construct is a plasmid, phage, phagemid,
The method according to claim 1, which is a viral vector or a cosmid. 27. The method of claim 26, wherein said expression construct is a shuttle vector capable of replicating in different host cell types. 28. The method of claim 1, wherein step (e) comprises purifying the complex. 29. The method of claim 4, wherein step (e) comprises purifying the complex. 30. The method of claim 3, wherein step (b) comprises purifying the complex. 31. The method according to claim 1, wherein the host cell is a bacterium, fungus, insect cell or animal cell. 32. The method of claim 2, 3, 4, or 6, wherein said host cell is a human cell. 33. The method according to claim 33, wherein the host cell is E. coli, S. cerevisiae, S. pombe, S. f.
rugiperda, COS cells, VERO cells, HeLa cells, Daudi cells, or CHO cells,
The method according to claim 1. 34. The method of claim 1, 2, 3, 4, 5, or 6, wherein the heat shock protein is selected from the group consisting of hsp70, hsp90, gp96, protein disulfide isomerase and combinations thereof. 35. The method according to claim 1, wherein the heat shock protein is hsp70. 36. The method according to claim 1, wherein the heat shock protein is hsp90. 37. The method according to claim 1, wherein the heat shock protein is gp96. 38. The method according to claim 1, wherein the heat shock protein is a protein disulfide isomerase. 39. The heat shock protein, wherein the host cell further comprises a nucleotide sequence encoding a heat shock protein or a fusion protein comprising a heat shock protein, wherein the nucleotide sequence is operably linked to a non-natural regulatory region. Or the method of claim 1 or 3, wherein the fusion protein is expressed by the host cell. 40. A method for eliciting an immune response in an individual, the method comprising:
Or administering to the individual a composition containing a heat shock protein complex non-covalently bound to a peptide in an immunogenic amount, which is prepared by the method according to 3, wherein the host cell and the individual The above method, wherein the species is different. 41. The method of claim 40, wherein said complex is substantially all or an uncharacterized portion thereof of a preselected hsp-containing peptide complex obtained from a host cell. 42. A method for treating or preventing cancer in an individual suffering from or desiring to prevent cancer, comprising binding non-covalently to a peptide prepared by the method of claim 1 or 2. The above method, which comprises administering to an individual an immunogenic composition containing the conjugate of the heat shock protein. 43. A method for treating a subject having cancer, comprising: (a) encoding a host cell containing a cDNA molecule prepared from an RNA molecule of a cancer tissue obtained from one or more human individuals by using the cDNA molecule; Culturing under conditions wherein the protein to be expressed is expressed by the host cell; (b) recovering from the host cell a complex of heat shock proteins non-covalently bound to one or more peptides; (c) administering the recovered complex to the subject. 44. The method of claim 43, wherein said cancerous tissue is obtained from one or more individuals different from said subject and having the same type of cancer as said subject. 45. The method of claim 43, wherein said cancerous tissue is obtained from said subject. 46. A method for preventing cancer in an individual who wants to prevent cancer, comprising: (a) using a host cell comprising a cDNA molecule prepared from an RNA molecule of a cancer tissue obtained from one or more human individuals; Culturing under conditions in which the host cell expresses the cDNA molecule to produce the encoded protein; (b) removing the heat shock protein non-covalently bound to one or more peptides from the host cell. Recovering the complex, and (c) administering the recovered complex to the individual. 47. The method of claim 43, wherein said subject is a human and said administered conjugate is purified. 48. A method for treating an individual with cancer, comprising: (a) isolating an RNA molecule from cancer cells obtained from the individual; and (b) producing a cDNA molecule from the RNA molecule. (C) introducing the cDNA molecule into one or more host cells in a form suitable for expression of the cDNA molecule; (d) transforming the host cell containing the cDNA molecule into a protein encoded by the cDNA molecule; Culturing under conditions expressed by the host cell; (e) recovering a complex of heat shock protein non-covalently bound to one or more peptides from the host cell; (f) recovering Administering the conjugate to the individual. 49. The cancer is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma , Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer,
Ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma , Liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, fetal carcinoma, vilmoma, cervical cancer, testis cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma , Medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia 48. The method of claim 43, 46 or 47, wherein the method is selected from the group consisting of: lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, and heavy chain disease. 50. The method of claim 43, 46 or 47, wherein the heat shock protein is selected from the group consisting of hsp70, hsp90, gp96, protein disulfide isomerase and combinations thereof.